Exposure to aerosol extract from heated tobacco products causes a drastic decrease of glutathione and protein carbonylation in human lung epithelial cells.

Heated tobacco products (HTPs) are an emerging class of tobacco goods that claim to have lower health risks than those of smoking combustible tobacco products. In this study, we exposed human lung epithelial cell lines to extracts prepared from HTP aerosols and combustible cigarette smoke to compare cytotoxicity. We focused on the effects of aldehydes present in the aerosols of HTPs at levels close to those in combustible cigarette smoke. Significant toxicity was confirmed for the HTP extract, albeit to a lesser extent than that with the combustible cigarette extract. When redox balance was evaluated by the oxidative loss of low-molecular-weight thiols in the cells, we found that total glutathione (GSH) contents and low-molecular-weight thiol levels were significantly decreased after exposure to the aerosol extract of HTPs. These results indicated that GSH is rapidly consumed during the detoxification of xenobiotics, such as aldehydes from tobacco extracts. Accordingly, exposure to the aerosol extract of HTPs resulted in the enhanced carbonylation of many proteins. In a simple comparison, the results for HTPs were significantly different from those obtained with combustible cigarette smoke, suggesting reduced toxicity of HTPs. However, we found significant and harmful effects after exposing lung epithelial cells to the aerosol extract of HTPs. Thus, a further comprehensive study is needed to clarify the lung damage induced via the long-term inhalation of aerosols from HTPs.

ACTIVATION OF HUMAN FIBROBLASTS BY CHRONIC RADIATION RATHER THAN ACUTE RADIATION.

Cancer-associated fibroblast (CAF), an activated type of fibroblast, is a major stromal cell that contributes to tumor initiation and development in the tumor microenvironment (TME). We previously reported that fractionated radiation rather than acute radiation causes progressive damage to mitochondria and increases the generation of reactive oxygen species, playing an important role in the fibroblast activation in normal tissue injury. Activated fibroblasts then become CAF by interacting with tumor cells, promoting tumor growth in vivo. We here examined the chronic radiation effect on fibroblast activation. Acute radiation (<2.5 Gy) did not increase alpha-Smooth muscle actin, a CAF marker expression in healthy human cells, whereas chronic radiation (2.5 Gy) did. It can be concluded that the induction of fibroblast activation changes across acute radiation, fractionated radiation, and chronic radiation depending on the irradiation technique. This study highlights that radiation activates fibroblasts, playing a role in radiation-related tumor development via TME formation.

Egg-jelly signal molecules for triggering the acrosome reaction in starfish spermatozoa.

It was in the early 1950s that J.C. Dan discovered the acrosome reaction in sea urchins, starfishes and several other marine invertebrates at Misaki Marine Biological Station on the Pacific coast of Japan. We now know that in many animals including mammals the acrosome reaction is an essential, and probably the most central, change in spermatozoa for fertilization. Starfish spermatozoa undergo the acrosome reaction upon encountering the jelly coat consisting of glycoproteins, steroid saponins, oligopeptides and inorganic components. To induce the acrosome reaction, three egg jelly components act in concert on the spermatozoa: a highly sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroidal saponins named Co-ARIS, and a group of glutamine-rich tetratriacontapeptides named sperm activating peptide (SAP). The action of ARIS is quite species-specific due to the specificity of ARIS-receptors in a restricted domain of the sperm surface and depends very much on sulfated saccharide chains. Co-ARIS is not much species-specific and its action depends on the sulfate group and steroid side chain. SAPs have a ring of 25 residues and increase the intracellular pH of spermatozoa. None of them can induce the acrosome reaction by itself in normal sea water, but ARIS does induce it in high Ca2+ or high pH sea water. Although a combination of ARIS and either Co-ARIS or SAP induces the acrosome reaction in normal sea water, all three are required to mimic the full activity of dissolved jelly coat.

Semiclassical quantization of strongly chaotic vibrations in an M7-like cluster.

We report semiclassical energy spectra of vibrational state of a cluster composed of seven identical atoms like Ar7 in terms of our previously developed semiclassical wave function, which we call the action-decomposed function. The classical dynamics of this vibrational state is strongly chaotic and undergoes a large amplitude motion due to structural isomerization, which demands a long run of trajectory calculation. Permutation of identical particles should also be taken into account as a quantum effect, since a single molecular shape can be shared by many permutational isomers. Furthermore, chaos causes a spurious divergence in the amplitude factor of a correlation function in the initial value representation, which arises from the amplitude factor (prefactor) of a semiclassical wave function, while the final-state representation is suffered from the well-known divergence arising at caustics. Both approaches therefore face tremendous difficulty in a long-time calculation of the correlation functions. We challenge to extract some limited number of spectral lines from such chaotic dynamics. We further apply a correlation function that is free of such a troublesome amplitude factor. Numerical results from all these schemes are reported.

Effects of glucagon-like peptide 1 analogue on the early phase of revascularization of transplanted pancreatic islets in a subcutaneous site.

OBJECTIVE: The subcutaneous space is an ideal site for pancreatic islet transplantation. However, one of the main obstacles is poor revascularization. Recently, glucagon-like peptide 1 (GLP-1) analogues are emerging as a new treatment option for patients with type 2 diabetes, because they have been shown to decrease ß-cell apoptosis. Therefore, we hypothesized that administration of a GLP-1 analogue in the early phase may facilitate revascularization of transplanted pancreatic islets by decreasing apoptotic changes of vascular endothelial cells within and without the graft. In this study, we evaluated the effects of GLP-1 analogue liraglutide on revascularization at a subcutaneous site with the use of a highly sensitive imaging system. We combined a dorsal skinfold chamber (DSC) technique with multiphoton laser-scanning microscopy (MPLSM). METHODS: Donor pancreatic islets isolated from C57BL/6-Tg (CAG-EGFP) mice were syngeneically transplanted into a dorsal skinfold chamber mounted on recipient mice. Male C57BL/6N mouse as recipients were divided into 3 groups: control, donor islet-treated, and recipient-treated groups. In the donor islet-treated group, the pancreatic islets were cultured with liraglutide (1 µmol/L) for 24 hours. The recipient-treated mice were injected with liraglutide (100 µg/kg subcutaneously) twice daily for 8 days. The time-dependent changes of newly formed vessels surrounding the islet grafts were imaged with MPLSM on days 1, 4, and 7. To evaluate islet graft revascularization, we measured vascular volume surrounding the islet with the Volocity system. RESULTS: In the first 4 days after pancreatic islet transplantation, no significant difference was detected in newly formed vessels among the 3 groups. Also, no significant difference was detected to increase rates at 7 days after transplantation. CONCLUSIONS: In this study, administration of GLP-1 analogue liraglutide in the early phase after pancreatic islet transplantation did not promote revascularization of transplanted islet grafts.

Assessment for revascularization of transplanted pancreatic islets at subcutaneous site in mice with a highly sensitive imaging system.

BACKGROUND: The subcutaneous space is one of the ideal sites for pancreatic islet transplantation, owing to the minimal invasiveness and easy access. However, the results of pancreatic islet transplantation in subcutaneous sites remain unsatisfactory. One of the main obstacles to successful pancreatic islet transplantation in subcutaneous sites is poor revascularization. Therefore, the aim of this study was to evaluate the revascularization process at subcutaneous sites with a highly sensitive imaging system combining a dorsal skinfold chamber (DSC) technique and multiphoton laser scanning microscopy (MPLSM). METHODS: A few pancreatic islets isolated from C57BL/6-Tg (CAG-EGFP) mice were syngeneically transplanted into nonmetallic DSCs mounted on the backs of C57BL/6J mice. Time-dependent changes in the newly formed vessels of pancreatic islets were imaged using MPLSM on days 1, 4, 7, 11, and 14 (n = 6). Texas Red was injected intravenously to visualize blood vessels. To evaluate islet graft revascularization, we measured vascular volume surrounding the islet using the Volocity system (Improvision). RESULTS: The percentages of vascular volume at days 1 and 14 were assumed to be 0 and 100%, respectively. The vascular volume on each day was 9.4 ± 6.5% (day 4), 34.9 ± 11.2% (day 7), and 21.1 ± 4.6% (day 11). CONCLUSIONS: The present study showed that a highly sensitive imaging system combining the DSC technique and MPLSM was a useful tool to analyze the revascularization process of pancreatic islets in a subcutaneous site.

Specific binding of acrosome-reaction-inducing substance to the head of starfish spermatozoa.

In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and species-specific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescein-isothiocyanate-labelled ARIS and 125I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact spermatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads.

Ultrastructural localization of acrosome reaction-inducing substance (ARIS) on sperm of the starfish Asterias amurensis.

Using colloidal gold tagged ligands we have identified the ultrastructural site of ARIS binding to intact and acrosome-reacted starfish sperm. In intact sperm, colloidal gold conjugated ARIS was specifically localized to a single domain (0.1-0.3 micron in diameter) on the plasma membrane. This site was located on the anterior-lateral aspect of the sperm head, that is, just peripheral to the region occupied by the acrosomal vesicle and periacrosomal components. When sperm were labeled with colloidal gold conjugated ARIS, washed to remove unbound label, and then induced to undergo the acrosome reaction, the labeled patch remained associated with the plasma membrane and was positioned just lateral to the acrosomal process. However, when sperm were suspended in labeled ARIS and induced to undergo the acrosome reaction, label was observed along the entire anterior aspect of the sperm head with the exception of the acrosomal process. Labeling along the entire anterior aspect of the sperm head in this case was deemed to be nonspecific and due to binding of colloidal gold tagged molecules to components formerly located within the acrosomal vesicle, as the same pattern was obtained using colloidal gold tagged bovine serum albumin. Quantitative and qualitative aspects of ARIS binding observed here by electron microscopy are in agreement with measured binding characteristics previously reported (Ushiyama et al., 1993a: Zygote 1:121-127; Ushiyama et al., 1993b: J Reprod Dev 39:53-54), and indicate that the site of labeled ARIS binding represents a specific plasma membrane domain occupied by ARIS receptors.

Estimation by radiation inactivation of the minimum functional size of acrosome-reaction-inducing substance (ARIS) in the starfish, Asterias amurensis.

In the starfish Asterias amurensis, the jelly coat of the eggs contains a glycoprotein essential for the induction of the acrosome reaction in homologous spermatozoa that is termed the acrosome-reaction-inducing substance (ARIS). ARIS is a highly sulphated and fucose-rich glycoprotein of extremely high molecular mass (> 10(4) kDa). ARIS was irradiated with high-energy electrons in order to estimate the minimum size required for its biological activity. The minimum functional unit or target size of ARIS was estimated to be c. 14 kDa by target size analysis. ARIS was significantly disintegrated by the irradiation, yet the total sugar content was not apparently reduced. The binding of 125I-labelled ARIS to spermatozoa competed with that of irradiated ARIS, although the affinity of ARIS was much reduced after irradiation.