[Consideration of Surgical Procedure for Repeated Pulmonary Metastases of Solitary Fibrous Tumor].

We report a case of a solitary fibrous tumor with repeated lung metastases after resection of the primary tumor. The patient was a 58-year-old man who had a left upper lobe lung tumor resected in 2018. The tumor was a solitary fibrous tumor arising from the visceral pleura. During the subsequent follow-up, the tumor repeatedly metastasized into the lung, and a total of three surgeries were performed. From the specimen at third surgery, the possibility of the enucleation of the tumor was speculated from the macroscopic and microscopic findings. Since repeated resections of the lung may cause the gradual deterioration of pulmonary function, the possibility of tumor enucleation will be discussed for the future treatment plan.

The Intravitreal Injection of Lanosterol Nanoparticles Rescues Lens Structure Collapse at an Early Stage in Shumiya Cataract Rats.

We designed an intravitreal injection formulation containing lanosterol nanoparticles (LAN-NPs) via the bead mill method and evaluated the therapeutic effect of LAN-NPs on lens structure collapse and opacification using two rat cataract models (SCR-N, rats with slight lens structure collapse; SCR-C, rats with the combination of a remarkable lens structure collapse and opacification). The particle size of lanosterol in the LAN-NPs was around 50-400 nm. A single injection of LAN-NPs (0.5%) supplied lanosterol into the lens for 48 h, and no irritation or muddiness was observed following repeated injections of LAN-NPs for 6 weeks (once every 2 days). Moreover, LAN-NPs repaired the slight collapse of the lens structure in SCR-N. Although the remarkable changes in the lens structure of SCR-C were not repaired by LAN-NP, the onset of opacification was delayed. In addition, the increase of cataract-related factors (Ca2+ contents, nitric oxide levels, lipid peroxidation and calpain activity levels) in the lenses of SCR-C was attenuated by the repeated injection of LAN-NPs. It is possible that a deficiency of lanosterol promotes the production of oxidative stress. In conclusion, it is difficult to improve serious structural collapse with posterior movement of the lens nucleus with a supplement of lanosterol via LAN-NPs. However, the intravitreal injection of LAN-NPs was found to repair the space and structural collapse in the early stages in the lenses.

Co-administration of Magnesium Ion Prevents Indomethacin-Induced Intestinal Ulcerogenic Lesions in Adjuvant-Induced Arthritis Rats.

In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.

Hesperetin prevents selenite-induced cataract in rats.

PURPOSE: This study investigated the ability of hesperetin, a natural flavonoid, to prevent selenite-induced cataracts in a rat model. METHODS: Animals were divided into four treatment groups: G1 (control group), G2 (hesperetin-treated group), G3 (selenite-induced cataract group), and G4 (hesperetin-treated selenite cataract group). Animals in the G1 and G3 groups were injected with vehicle alone, while those in the G2 and G4 groups received a subcutaneous injection of hesperetin (0.4 µg/g bodyweight on days 0, 1, and 2, corresponding to P13, P14, and P15). Sodium selenite (20 µmol/g bodyweight given 4 h after the hesperetin injection on day 0) was administered to rats in the G3 and G4 groups to induce cataract formation. Lenses were observed with slit-lamp microscopy, and filensin degradation and the decreased glutathione (GSH) and ascorbic acid levels in the lens were measured on day 6. RESULTS: Lenses in the G3 group showed mature central opacity, while some lenses in the G4 group lacked central opacity and had lower-grade cataracts. All lenses in the G1 and G2 groups were transparent. Expression of the 94 kDa and 50 kDa forms of filensin was significantly decreased in the lenses in the G3 group compared with those in the G1 and G2 groups. Interestingly, these forms of filensin rescued the rat lenses in the G4 group. In the G3 group lenses, the GSH and ascorbic acid levels were lower than in the control group but were normalized in the G4 group lenses. CONCLUSIONS: The results suggest that hesperetin can prevent selenite-induced cataract formation.

Effects of truncations in the N- and C-terminal domains of filensin on filament formation with phakinin in cell-free conditions and cultured cells.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification.

Lung adenocarcinoma and squamous cell carcinoma difficult for immunohistochemical diagnosis can be distinguished by lipid profile.

In patients with unresectable non-small cell lung cancer, histological diagnosis is frequently based on small biopsy specimens unsuitable for histological diagnosis when they are severely crushed and do not retain their morphology. Therefore, establishing a novel diagnostic method independent of tissue morphology or conventional immunohistochemistry (IHC) markers is required. We analyzed the lipid profiles of resected primary lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC) specimens using liquid chromatography-tandem mass spectrometry. The specimens of 26 ADC and 18 SQCC cases were evenly assigned to the discovery and validation cohorts. Non-target screening on the discovery cohort identified 96 and 13 lipid peaks abundant in ADC and SQCC, respectively. Among these 109 lipid peaks, six and six lipid peaks in ADC and SQCC showed reproducibility in target screening on the validation cohort. Finally, we selected three and four positive lipid markers for ADC and SQCC, demonstrating high discrimination abilities. In cases difficult to diagnose by IHC staining, [cardiolipin(18:2_18:2_18:2_18:2)-H]- and [triglyceride(18:1_17:1_18:1) + NH4]+ showed the excellent diagnostic ability for ADC (sensitivity: 1.00, specificity: 0.89, accuracy: 0.93) and SQCC (sensitivity: 0.89, specificity: 0.83, accuracy: 0.87), respectively. These novel candidate lipid markers may contribute to a more accurate diagnosis and subsequent treatment strategy for unresectable NSCLC.

Retention index of FDG-PET/CT SUVmax of the primary tumor in non-small cell lung cancer as a predictor of lymph node metastasis: a retrospective study.

BACKGROUND: Accurate staging of non-small cell lung cancer is key in treatment planning and prediction of prognosis. We investigated the correlation between the maximum standardized uptake value (SUVmax) retention index (RI) of the primary tumor and lymph node metastasis in non-small cell lung carcinoma. We also evaluated the tendencies according to the histological types. METHODS: We retrospectively evaluated 218 non-small cell lung cancer (NSCLC) tumors from 217 patients who underwent preoperative fluorodeoxyglucose-positron emission tomography/computed tomography (PET/CT) followed by lung surgery and lymph node resection between July 2015 and August 2020. All primary tumors were calculated as the SUVmax at 50 min (SUVmaxearly [SUVmaxe]) and 120 min (SUVmaxdelayed [SUVmaxd]), and RI. The clinicopathological factors of interest were compared based on lymph node metastasis status and NSCLC histopathological subtype. RESULTS: The median SUVmaxe and SUVmaxd of the primary tumors were 3.3 and 4.2, respectively, and the median RI was 0.25. The RI was significantly higher in the pN(+) (n = 44) group (0.30) compared to the pN0 (n = 174) group (0.24) (p = 0.01). In patients with adenocarcinoma (n = 145), the RI was also significantly higher in the pN(+) (n = 29) group (0.29) compared to the pN0 (n = 116) group (0.16) (p < 0.01). A high RI of the primary tumor was an independent risk factor for lymph node metastasis, particularly in patients with adenocarcinoma (odds ratio: 12.30, p < 0.05). CONCLUSIONS: The RI of primary NSCLC tumors can help predict lymph node metastases, particularly in patients with adenocarcinoma.

Placement of KANI® plate inside the chest wall for rib fixation: Prevention for organ injuries caused by crossed rib edges and plate claws.

The claw-type titanium plate has been successfully applied to manage a flail chest. However, rare and life-threatening organ injury occurs due to an insufficient claw bend. We report an ingenuity of surgical fixation using KANI® plates (USCI Japan, Tokyo, Japan) in a flail chest. A 60-year-old man with a severe flail chest underwent a surgical rib fixation. He had multiple rib fractures accompanied by dislocation and protruding crossed rib edges; we assumed a possibility of lung injury during a standard procedure in which the KANI® plates would be placed from outside the chest wall. Therefore, we placed KANI® plates inside the chest wall to ensure sufficient claw bend and to cover crossed rib edges to prevent organ injuries. We propose that our new ingenuity provides a safe and tight rib fixation in rib fractures with protruding crossed rib edges which the standard method cannot flatten.

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens.

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 µM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.

The effect of the interaction between aquaporin 0 (AQP0) and the filensin tail region on AQP0 water permeability.

PURPOSE: To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lens-specific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated. METHODS: Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOH-terminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOH-terminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin. RESULTS: The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lens AQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0. CONCLUSIONS: The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens.

Instillation of Ophthalmic Formulation Containing Nilvadipine Nanocrystals Attenuates Lens Opacification in Shumiya Cataract Rats.

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

Characterization and localization of side population cells in the lens.

PURPOSE: Side population (SP) cells were isolated and the possibility whether lens epithelial cells contain stem cells was investigated. METHODS: Mouse lens epithelial cells were stained by Hoechst 33342 and then sorted by fluorescence-activated cell sorting (FACS). The expression of stem cell markers in sorted SP cells and the main population of epithelial cells were analyzed by quantitative real-time PCR. Localization of SP cells in the mouse lens was studied by fluorescence microscopy. RESULTS: The sorted cells contained SP cells, which were no longer separable by FACS following treatment with verapamil. The number of SP cells decreased with aging, but the adult mouse lens still contained SP cells. Phase contrast microscopy revealed that SP cells were smaller in size than non-SP cells. SP cells were localized near the equator region in lens epithelial cell layers. SP cells expressed higher levels of the stem cell markers ATP-binding cassette transporter G2 (ABCG2), p75 neurotrophin receptor (p75NTR), nestin (nes), B-cell lymphoma 2 (Bcl2), and cell surface antigen Sca-1 mRNA than the main population cells. These results suggest that SP cells contain a high proportion of stem cells. CONCLUSIONS: The SP cells in the lens contain stem cells, and these stem cells are localized around the germinative zone.

Ophthalmic In Situ Gelling System Containing Lanosterol Nanoparticles Delays Collapse of Lens Structure in Shumiya Cataract Rats.

We attempted to prepare ophthalmic in situ gel formulations containing lanosterol (Lan) nanoparticles (LA-NPs/ISG) and investigated the characteristics, delivery pathway into the lens, and anti-cataract effects of LA-NPs/ISG using SCR-N (rats with slight lens structure collapse) and SCR-C (rats with a combination of remarkable lens structure collapse and opacification). LA-NPs/ISG was prepared by bead milling of the dispersions containing 0.5% Lan powder, 5% 2-hydroxypropyl-ß-cyclodextrin, 0.5% methylcellulose, 0.005% benzalkonium chloride, and 0.5% mannitol. The particle size distribution of Lan was 60-250 nm. The LA-NPs/ISG was gelled at 37 °C, and the LA-NPs/ISG was taken into the cornea by energy-dependent endocytosis and then released to the intraocular side. In addition, the Lan contents in the lenses of both SCR-N and SCR-C were increased by the repetitive instillation of LA-NPs/ISG (twice per day). The space and structure collapse in the lens of SCR-N with aging was attenuated by the instillation of LA-NPs/ISG. Moreover, the repetitive instillation of LA-NPs/ISG attenuated the changes in cataract-related factors (the enhancement of nitric oxide levels, calpain activity, lipid peroxidation levels, Ca2+ contents, and the decrease of Ca2+-ATPase activity) in the lenses of SCR-C, and the repetitive instillation of LA-NPs/ISG delayed the onset of opacification in the SCR-C. It is possible that the LA-NPs/ISG is useful in maintaining lens homeostasis.

The function of filensin and phakinin in lens transparency.

PURPOSE: Beaded filaments are lens cell-specific intermediate filaments composed of two proteins: filensin and phakinin (CP49). Filensin and phakinin are believed to function in the maintenance of lens transparency. To elucidate the function of filensin and phakinin at the molecular level, we examined the degradation of these two proteins in normal and cataractous rat lenses. METHODS: A hereditary cataract model, the Shumiya cataract rat (SCR), was used for these studies. Anti-filensin antibodies were raised against three different regions of the protein, the rod domain, the inner region of the tail domain, and the outer region of the tail domain. Anti-filensin and anti-phakinin antibodies were used to examine the conformation of degradation of filensin and phakinin by western blot analysis and fluorescent immunocytochemistry of cryosectioned lenses. RESULTS: In the normal lens, filensin was processed from a 94 kDa protein to proteins of 50 kDa and 38 kDa. Similarly, phakinin was processed from a 49 kDa protein to one of 40 kDa. The concentrations of filensin and phakinin in the rat lens cortex fluctuated with age and decreased during cataractogenesis. The 50 kDa form of filensin decreased significantly before opacification. In the normal lens, phakinin, the filensin rod domain, and the filensin inner tail domain localized to membrane lining regions in the shallow cortex and to the central region of the cytoplasm in the deep cortex. The COOH-terminal domain of filensin localized to the membrane lining region in the deep cortex. In pre-cataractous lenses, phakinin and the filensin rod domain localized primarily to the membranes lining the shallow cortex region and were distributed throughout the cytoplasm of lens fiber cells in the deep cortex. CONCLUSIONS: The 50 kDa form of filensin is important for the localization of beaded filaments in lens fiber cells and for lens transparency.

NADH photo-oxidation is enhanced by a partially purified lambda-crystallin fraction from rabbit lens.

PURPOSE: In the rabbit lens, high levels of reduced nicotinamide adenine dinucleotide (NADH) can function as a near-ultraviolet light (near-UV) filter, an effect apparently achieved by specific nucleotide binding to lambda-crystallin. The present investigation asks whether lambda-crystallin enhances NADH photo-oxidation by superoxide radicals produced via a photosensitization reaction of near-UV with NADH. METHODS: Lambda-crystallin was partially purified from rabbit lens soluble fraction by a two-step gel filtration and affinity column chromatography procedure. NADH solutions with or without partially purified lambda-crystallin were subjected to near-UV irradiation or exposed to superoxide generated enzymatically by the xanthine/xanthine oxidase system. NADH oxidation was determined by assaying the decrease of absorbance at 340 nm. RESULTS: When irradiated with near-UV, free NADH was oxidized very little in the absence of lambda-crystallin. In contrast, NADH photo-oxidation was rapidly initiated in the presence of partially purified lambda-crystallin. This lambda-crystallin-enhanced NADH photo-oxidation was totally inhibited by adding superoxide dismutase. We also found that lambda-crystallin largely increased NADH oxidation by a superoxide that is generated enzymatically. These results indicate that NADH bound to lambda-crystallin rapidly reacts with superoxides. The reactivity of bound NADH with superoxide was almost equivalent to that of ascorbic acid. However, lambda-crystallin-enhanced NADH oxidation exceeded the superoxide levels generated by NADH photosensitization and xanthine/xanthine oxidase. CONCLUSIONS: We conclude that NADH binding to lambda-crystallin enhances NADH photo-oxidation through a radical chain reaction mechanism that is initiated by superoxides generated by NADH photosensitization and propagated by another superoxide from the molecule oxygen. High concentrations of NADH bound to lambda-crystallin may be beneficial to the rabbit lens in scavenging the low amounts of superoxide produced by near-UV absorption, since oxygen tension in the lens is very low. We also discuss the function of near-UV-filtering and the anti-photo-oxidation systems in other vertebrate lenses.

Model for Studying Anti- Allergic Drugs for Allergic Conjunctivitis in Animals.

Allergic conjunctivitis (AC), which is characterized by ocular itching, hyperemia, and edema, deteriorates quality of life. In this study, effects of anti-allergic drugs were evaluated by assessing eye-scratching behavior, the number of eosinophils in conjunctiva epithelial tissues, and concentrations of chemical mediators in the tears of the guinea pig model of ovalbumin (OA)-induced AC. METHODOLOGY: On day 0, 3-week-old guinea pigs were sensitized by OA subconjunctival injections. On days 15, 17, and 19, OA solution was administered. Anti-allergic eye drops were administered 5 and 15 min before the final OA challenge on day 19. Scratching behavior within 1 h after OA exposure was studied. Eosinophils in the conjunctiva were stained with Giemsa reagent. Histamine and substance P (SP) concentrations in tears were measured using ELISA. RESULTS: Subconjunctivally injected guinea pigs were observed for clinical symptoms. Scratching responses significantly reduced with ketotifen or olopatadine treatment. Eosinophil numbers reduced in animals treated with ketotifen, levocabastine, or tranilast. Histamine and/or SP concentrations in tears were inhibited by some of these anti-allergic drugs. CONCLUSIONS: It is important to assess the anti-allergic AC drugs objectively because there are several of these drugs currently available. This model allows for an objective evaluation of anti-allergic drugs for AC.

The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0.

PURPOSE: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion. METHODS: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume. RESULTS: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability. CONCLUSIONS: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

NADH binding properties of rabbit lens lambda-crystallin.

PURPOSE: The present investigation aims to evaluate the NADH binding ability of lambda-crystallin, a taxon-specific enzyme-crystallin, in the rabbit lens. METHODS: A lambda/betaL1-crystallin fraction was separated from the rabbit lens soluble fraction by gel filtration and the enzyme-crystallin was partially purified by subsequent affinity column chromatography. Analysis of NADH bound to the lambda-crystallin preparation was performed using spectrophotometric and enzymological methods. Binding of added NADH to the enzyme-crystallin preparation was also analyzed using a simple ultrafiltration method, which was theoretically equivalent to equilibrium dialysis, to study additional NADH binding to the protein. RESULTS: The prepared lambda-crystallin samples clearly exhibited an absorption maximum at 340 nm, even though they were thoroughly dialyzed. This was due to the presence of nondialyzable NADH bound tightly to the protein. The bound NADH was removed by charcoal treatment, and extracted by 0.1% SDS or 70 degrees C heat treatment. A dissociation constant (Kd) of less than 5 nM indicated tight binding of NADH. The quantity of bound NADH in the 88% purified 33 kDa enzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. Additional looser binding of added NADH to lambda-crystallin was observed in both the lambda/betaL1-crystallin fraction (including the full-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which might be generated by cleavage of the 33 kDa protein: 64%) and the partially purified enzyme-crystallin. It was assumed from the analysis of binding titration that some (about 30%) of the 33 kDa protein and most of the lower molecular weight proteins still possessed the ability to loosely bind NADH. Kd values of their lower affinity binding were determined to be 2 or 6 microM. CONCLUSIONS: From the present study, we conclude that lambda-crystallin plays a sufficiently important role as a NADH binding protein to maintain high levels of this nucleotide in the rabbit lens.

Effect of hesperetin on chaperone activity in selenite-induced cataract.

BACKGROUND: Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. METHODOLOGY: Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. RESULTS: G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. CONCLUSIONS: Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite.

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte: (meiosis/maturation/immunofluorescence/microtubule/spindle).

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.