Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.     

Dosimetric selection for helical tomotherapy based stereotactic ablative radiotherapy for early-stage non-small cell lung cancer or lung metastases

Background

No selection criteria for helical tomotherapy (HT) based stereotactic ablative radiotherapy (SABR) to treat early stage non-small cell lung cancer (NSCLC) or solitary lung metastases has been established. In this study, we investigate the dosimetric selection criteria for HT based SABR delivering 70 Gy in 10 fractions to avoid severe toxicity in the treatment of centrally located lesions when adequate target dose coverage is desired.

Materials and Methods

78 HT-SABR plans for solitary lung lesions were created to prescribe 70 Gy in 10 fractions to the planning target volume (PTV). The PTV was set to have ≥95% PTV receiving 70 Gy in each case. The cases for which dose constraints for ≥1 OAR could not be met without compromising the target dose coverage were compared with cases for which all target and OAR dose constraints were met.

Results

There were 23 central lesions for which OAR dose constraints could not be met without compromising PTV dose coverage. Comparing to cases for which optimal HT-based SABR plans were generated, they were associated with larger tumor size (5.72±1.96 cm vs. 3.74±1.49 cm, p<0.0001), higher lung dose, increased number of immediately adjacent OARs ( 3.45±1.34 vs. 1.66±0.81, p<0.0001), and shorter distance to the closest OARs (GTV: 0.26±0.22 cm vs. 0.88±0.54 cm, p<0.0001; PTV 0.19±0.18 cm vs. 0.48±0.36 cm, p = 0.0001).

Conclusion

Delivery of 70 Gy in 10 fractions with HT to meet all the given OAR and PTV dose constraints are most likely when the following parameters are met: lung lesions ≤3.78 cm (11.98 cc), ≤2 immediately adjacent OARs which are ≥0.45 cm from the gross lesion and ≥0.21 cm from the PTV.     

The pigment stoichiometry in a chlorophyll a/c type photosynthetic antenna

The trimeric fucoxanthin-chlorophyll a/c protein (FCP) was purified from a Japanese brown alga, Cladosiphon okamuranus TOKIDA. Its pigment stoichiometry was determined to be chlorophyll (Chl) a:Chl c (1):Chl c (2):fucoxanthin?=?4.6:1.1:1.0:5.5 by a combination of binary HPLC and (1)H NMR spectroscopy. No violaxanthin found bound to the FCP. The ratio of Chl c/Chl a in this FCP is amongst the highest so far reported.     

Polarization angle dependence of stark absorption spectra of spirilloxanthin bound to the reconstituted LH1 complexes using LH1-subunits isolated from the purple photosynthetic bacterium Rhodospirillum rubrum

Reconstituted LH1 complexes were prepared using the LH1 subunit-type complexes, isolated from the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum, and purified all-trans spirilloxanthin. Stark absorption spectra of spirilloxanthin bound to both the native and reconstituted LH1 complexes were compared in different polarization angles (χ) against the external electric field. From the polarization angle dependence of the Stark absorption spectra, two angles were determined in reference to the direction of transition dipole moment (m) of spirilloxanthin: one is the change in polarizability upon photoexcitation (Δα), θ(Δα) and the other is the change in static dipole moment upon photoexcitation (Δμ), θ(Δμ). Despite the symmetric molecular structure of all-trans spirilloxanthin, its Stark absorption spectra show pronounced values of Δμ. This large Δμ values essentially caused by the effect of induced dipole moment through Δα both in the cases for native and reconstituted LH1 complexes. However, slightly different values of θ(Δα) and θ(Δμ) observed for the native LH1 complex suggest that spirilloxanthin is asymmetrically distorted when bound to the native LH1 complex and gives rise to intrinsic Δμ value.     

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature-labile and had an optimal pH of 7.5. Metallo-protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability-enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability-enhancing activity in skin. This suggests that a trypsin-like protease from skin mediates the activity of the protease to enhance its vascular permeability.     

Effects of Temperature on the Circadian Conidiation Rhythm of Temperature-Sensitive Mutants of Neurospora crassa

Period lengths at different temperatures and phase responsecurves at a high temperature (35°C) of circadian conidiationrhythms were examined in 13 temperature-sensitive (un) strainsof Neurospora crassa. Two strains, un-16 and un-18, had longerperiod lengths than the wild-type strain even at permissivetemperatures. Period lengths of six strains, un-4, un-11, un-16,un-18, un-19 and un-22, changed differently from that of thewild-type strain at restrictive temperatures. However, the shapeof phase response curves for high temperature (35°C) for3 h was almost the same for all un strains and the wild-typestrain. We isolated 97 temperature-sensitive mutants with periodlengths from 19.2 to 24.8 h and determined the dependence ontemperature of the period length of the conidiation rhythm foreach mutant. The mutants could be divided into four differentgroups in terms of their responses to changes in temperature. (Received September 8, 1993; Accepted March 10, 1994)     

Citrus limonoid by-products as insect control agents

Limonoids are a group of chemically related bitter tetranortriterpene derivatives found predominantly in Rutaceae and Meliaceae plants (Ourison et al., 1964). Interest in the Rutaceae limonoids has centered around limonoid removal from consumable citrus products. For example, bitterness in citrus juices (as well as in other citrus products) due to limonoids has become an increasingly serious economic problem (Wilson & Crutchfield, 1968; Sinclair, 1972). Interest in the Meliaceae limonoids, on the other hand, has centered on their efficacy as pest control and/or antitumor agents (Kubo & Klocke, 1981, 1982; Nakanishi, 1977, 1980). For example, azadirachtin, isolated from several Meliaceae trees, has proven to be a potent natural product against a myriad of insect and nematode pests (Warthen, 1979). In fact, we have isolated azadirachtin from the fresh fruit of Azadirachta indica as a potent insect ecdysis inhibitor against four agricultural pest insects with artificial diet feeding assay (Kubo & Klocke, in litt).     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non-treated ones by 2-D PAGE in the pI range of 4.0-7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI-TOF/TOF MS and subsequent protein database searching. They included unc-18 protein homolog 67K, collapsin response mediator protein (CRMP)-2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post-translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.