The impact of oocyte death on mouse primordial follicle formation and ovarian reserve.

Background: Ovaries, the source of oocytes, maintain the numbers of primordial follicles, develop oocytes for fertilization and embryonic development. Although it is well known that about two-thirds of oocytes are lost during the formation of primordial follicles through cyst fragmentation and the aggregation of oocytes within the cyst, the mechanism responsible for this remains unclear. Methods: We provide an overview of cell death that is associated with the oocyte cyst breakdown and primordial follicle assembly along with our recent findings for mice that had been treated with a TNFα ligand inhibitor. Main Findings: It is generally accepted that apoptosis is the major mechanism responsible for the depletion of germ cells. In fact, a gene deficiency or the overexpression of apoptosis regulators can have a great effect on follicle numbers and/or fertility. Apoptosis, however, may not be the only cause of the large-scale oocyte attrition during oocyte cyst breakdown, and other mechanisms, such as aggregation, may also be involved in this process. Conclusion: The continued study of oocyte death during primordial follicle formation could lead to the development of novel strategies for manipulating the primordial follicle pool, leading to improved fertility by enhancing the ovarian reserve.

Retention of higher fertility depending on ovarian follicle reserve in cystine-glutamate transporter gene-deficient mice.

The cystine-glutamate transporter (xCT) is responsible for the transport of cystine into cells. We recently found that xCT-deficient (xCTKO) aged mice maintained a higher rate of ovulation and ovarian weight compared with wild-type (WT) mice. It has been reported that a xCT deficiency in cultured cells induces autophagy through the suppression of mTOR survival pathways. We have previously reported that starvation in neonatal mice increases the number of primordial follicles with concomitant autophagy activation. Therefore, we investigated age-related changes in follicle reserve and fertility in xCTKO mice and clarified whether the PI3K/AKT/mTOR signaling pathway contributes to this. The numbers of offspring in the xCTKO mice aged 10 and 12 months were significantly higher than those in the WT mice. The primordial follicle numbers in xCTKO neonatal mice tended to be higher than WT mice during all times evaluated. In contrast, the primary follicle number was significantly lower in the xCTKO mice at 60 h after birth. The expression of p-AKT, which promotes follicle development, was significantly lower in xCTKO mice than that in WT mice, whereas the expression ratios of LC3-II/LC3-I were significantly higher. The xCTKO mice had significantly more primordial follicles than WT mice at 2 months of age and showed a similar trend at 13-15 months of age. These results suggest that the maintenance of fertility in aged xCTKO mice can be attributed to high follicle reserve after puberty by suppression of follicle activation during the neonatal period.

An ectopic thymoma arising in the middle mediastinum that was difficult to distinguish from a lymph node metastasis.

BACKGROUND: Ectopic thymomas often occur in the upper mediastinum; however, they rarely arise in the middle mediastinum, especially on the dorsal side of the innominate vein and superior vena cava in the peribronchial region. CASE PRESENTATION: Six years prior, a 27-year-old female presented to our department and was diagnosed with locally advanced left breast cancer. First, we administered chemotherapy including an anti-human epidermal growth factor receptor 2 antibody. The size of the tumor was markedly reduced, and a radical operation involving mastectomy and axillary lymph node dissection was then performed. The patient underwent radiotherapy after the mastectomy, followed by trastuzumab therapy; she continued to receive endocrine therapy thereafter. She underwent computed tomography once a year after the surgery, and a nodule in the middle mediastinum on the dorsal side of the innominate vein and superior vena cava in the parabronchial region was detected at 4 years. We speculated that the nodule was a solitary mediastinal lymph node metastasis from her breast cancer; therefore, we performed thoracoscopic resection of the tumor. We diagnosed the tumor as a thymoma. Currently, the patient visits our hospital to receive continuous hormone therapy for her breast cancer, and the latest computed tomography scan demonstrated no metastases from or recurrence of her breast cancer or thymoma. CONCLUSIONS: We report a case of ectopic thymoma in the middle mediastinum. The tumor, which was detected during systemic therapy for locally advanced breast cancer, was located on the dorsal side of the innominate vein and superior vena cava in the parabronchial region and was indistinguishable from a lymph node metastasis from breast cancer.

Feasibility of autologous fibrin glue in general thoracic surgery.

BACKGROUND: Fibrin glue effectively controls air leakage in lung surgery; however, allogenic fibrin glue cannot eliminate the risks of infection and allergy despite current sterilization methods. Autologous fibrin glue (AFG) could be a good alternative, but is not commonly used worldwide because of its limited availability and lack of evidence. Herein, we report clinical outcomes of AFG in thoracic surgery. METHODS: We retrospectively analyzed patients who underwent lobectomies or segmentectomies between November 2016 and September 2017 in our institution. We used two types of AFGs. One was a partially-autologous fibrin glue (PAFG), the components of which are largely autologous but which contains allogenic thrombin. The other was a completely-autologous fibrin glue (CAFG) which has no allogenic components. PAFG was used in the first half of the study period, after which CAFG was used from March 2017 onward. Patients who did not undergo AFG generation were categorized as the non-AFG group. The perioperative outcomes of the three groups were evaluated. RESULTS: A total of 207 patients underwent lung surgery, including 118 lobectomies and 89 segmentectomies. Among them, 83 patients received PAFG, 94 received CAFG, and 30 received non-AFG. The mean postoperative drainage period was within a few days in each group (PAFG vs. CAFG vs. non-AFG: 3.23±3.91 vs. 3.16±4.04 vs. 3.17±4.16 days, respectively; P=0.405), and the incidence of postoperative prolonged air leakage was within an acceptable range (PAFG vs. CAFG vs. non-AFG: 13.3% vs. 12.8% vs. 16.7%, respectively; P=0.821). CONCLUSIONS: The use of AFG is clinically feasible for patients who undergo lobectomies or segmentectomies. AFGs could be a viable alternative to conventional allogenic fibrin glues.

Ascorbic acid and CoQ10 ameliorate the reproductive ability of superoxide dismutase 1-deficient female mice†.

Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44-75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

A heterozygous deficiency in protein phosphatase Ppm1b results in an altered ovulation number in mice.

Ppm1b, a metal­dependent serine/threonine protein phosphatase, catalyzes the dephosphorylation of a variety of phosphorylated proteins. Ppm1b­/­ mouse embryos die at the fertilized oocyte stage, whereas Ppm1b+/­ mice with a C57BL/6 background exhibit no phenotypic abnormalities. Because the C57BL/6 strain produces a limited number of pups, in an attempt to produce Ppm1b­/­ mice, congenic Ppm1b+/­ mice with an ICR background were established, which are more fertile and gave birth to more pups. As a result, however, no Ppm1b­/­ offspring were obtained when pairs of Ppm1b+/­ ICR mice were bred again. Ppm1b+/­ male and female ICR mice were analyzed from the viewpoint of fecundity. The Ppm1b haploinsufficiency had no effect on testicular weight or the number of sperm in male mice. Despite the fact that the levels of Ppm1b protein in the ovaries of sexually mature Ppm1b+/­ mice were decreased compared with those of Ppm1b+/+ mice, there appeared to be no significant difference in the histological appearance of the ovaries, litter sizes or plasma progesterone levels at the estrous stage. When superovulation was induced by stimulation using a hormone treatment, the number of ovulated oocytes were the same for Ppm1b+/­ and Ppm1b+/+ mice at 4 weeks of age when the estrous cycle did not proceed, however, the number of ovulated oocytes was lower in sexually mature Ppm1b+/­ mice at 11 weeks of age compared with Ppm1b+/+ mice in the first and the second superovulation cycles. These collective results suggest that follicle development is excessive in Ppm1b+/­ mice, and that this leads to a partial depletion of matured follicles and a corresponding decrease in the number of ovulated oocytes.

Simultaneous three-port thoracoscopic surgery for bilateral lung cancers with a pulmonary vein anomaly: A case report.

INTRODUCTION: Simultaneous resection of bilateral lung cancers is technically challenging but may be preferable to a staged procedure in patients with a partial anomalous pulmonary venous connection (PAPVC) in an affected lobe. We performed single-stage resection of bilateral lung cancers in a patient with a PAPVC. PRESENTATION OF CASE: A 73-year-old man was diagnosed as having bilateral lung cancers (right, cT3N1M0, stage IIIA and left, cT2aN0M0, stage IB). Left upper trisegmentectomy was performed, followed by right upper lobectomy with deep wedge bronchoplasty. A PAPVC was found incidentally in the affected right upper lobe and successfully divided. The postoperative course was uneventful and he commenced chemoradiotherapy. DISCUSSION: Resection of the PAPVC, which was located in the same lobe as the lung cancer, would have mitigated load increase in the right heart and may have alleviated the adverse effects of bilateral lung resection. Moreover, the single-stage procedure likely shortened the overall duration of treatment. CONCLUSION: Single-stage bilateral thoracoscopic resection may have advantages over staged procedures in some patients with PAPVCs.

Decreased reproductive performance in xCT-knockout male mice.

Sulphoxidation occurs in protamines that are enriched in cysteine and supplies chromatin for packaging. The extracellular fluid contains higher levels of oxidised cysteine (cystine), and some cells utilise system xc-, a cystine transporter in which xCT is the main protein component, to fulfil the need for cysteine. We hypothesised that system xc- might ensure the supply of cysteine needed for spermatogenesis. The reproductive ability of xCT-/- male mice at 6- to 18-weeks of age appeared to be lower than xCT+/+ male mice. The courtship behaviour of the xCT-/- male mice was undynamic, which appeared to be associated with the low reproductive ability of xCT-/- male mice. xCT was found to be expressed in mouse testes, notably in Sertoli cells, as well as in the epididymis and the levels were increased at the time of sexual maturation. Despite the normal histological appearance of testicular tissues, the cauda epididymis of xCT-/- mice contained round, greater numbers of immature spermatogenic cells than that of xCT+/+ mice. However, there were no significant differences in the numbers of sperm stored in the cauda epididymis or in the concentrations of cysteine or glutathione in the testes. The resulting sperm had normal fertilising ability. Thus, system xc- appears to function as a backup system for supplying cysteine to testes and play a pivotal role in supplying cysteine for normal sexual behaviour by a mechanism that is different from that for the supply of cysteine in spermatogenesis.

Pulmonary Neoplasms in Patients with Birt-Hogg-Dubé Syndrome: Histopathological Features and Genetic and Somatic Events.

Birt-Hogg-Dubé syndrome (BHD) is an inherited disorder caused by genetic mutations in the folliculin (FLCN) gene. Individuals with BHD have multiple pulmonary cysts and are at a high risk for developing renal cell carcinomas (RCCs). Currently, little information is available about whether pulmonary cysts are absolutely benign or if the lungs are at an increased risk for developing neoplasms. Herein, we describe 14 pulmonary neoplastic lesions in 7 patients with BHD. All patients were confirmed to have germline FLCN mutations. Neoplasm histologies included adenocarcinoma in situ (n = 2), minimally invasive adenocarcinoma (n = 1), papillary adenocarcinoma (n = 1), micropapillary adenocarcinoma (n = 1), atypical adenomatous hyperplasia (n = 8), and micronodular pneumocyte hyperplasia (MPH)-like lesion (n = 1). Five of the six adenocarcinoma/MPH-like lesions (83.3%) demonstrated a loss of heterozygosity (LOH) of FLCN. All of these lesions lacked mutant alleles and preserved wild-type alleles. Three invasive adenocarcinomas possessed additional somatic events: 2 had a somatic mutation in the epidermal growth factor receptor gene (EGFR) and another had a somatic mutation in KRAS. Immunohistochemical analysis revealed that most of the lesions were immunostained for phospho-mammalian target of rapamycin (p-mTOR) and phospho-S6. Collective data indicated that pulmonary neoplasms of peripheral adenocarcinomatous lineage in BHD patients frequently exhibit LOH of FLCN with mTOR pathway signaling. Additional driver gene mutations were detected only in invasive cases, suggesting that FLCN LOH may be an underlying abnormality that cooperates with major driver gene mutations in the progression of pulmonary adenocarcinomas in BHD patients.

Long-term tear volume changes after blepharoptosis surgery and blepharoplasty.

PURPOSE: To evaluate long-term changes in tear volume by using video meniscometry following blepharoptosis surgery and upper blepharoplasty. METHODS: Forty-three eyes of 27 patients with blepharoptosis and 29 eyes of 18 patients with dermatochalasis without lacrimal duct obstruction or other eyelid diseases underwent anterior approach levator advancement or blepharoplasty. Tear volume was evaluated by measurement of tear meniscus radius (R), using video meniscometry preoperatively and at 1.5, 3, and 6 months postoperatively. Margin reflex distance-1 (MRD-1) was measured before and after surgery by using photographs. RESULTS: After blepharoptosis surgery, the mean MRD-1 was significantly increased: 0.45 mm preoperatively, 3.64 mm at 1.5 months, 3.56 mm at 3 months, and 3.57 at 6 months postoperatively (P < 0.001), and the average R value was significantly decreased: 0.29 mm preoperatively, 0.22 mm at 1.5 months, 0.23 mm at 3months, and 0.24 mm at 6 months postoperatively (P < 0.05). Preoperative R was significantly correlated to the reduction rate of R (ΔR). A higher preoperative R was more likely to be decreased (P < 0.01). Postoperative MRD-1 and change in MRD-1 were not correlated to ΔR. After blepharoplasty, the preoperative mean MRD-1 (3.11 mm) was significantly decreased at 1.5 months (2.47 mm; P < 0.01) and 3 months (2.71 mm; P < 0.05) but recovered at 6 months (3.14 mm). However, the average R was not changed: 0.31 mm preoperatively, 0.34 mm at 1.5 months, 0.31 mm at 3 months, and 0.33 mm at 6 months postoperatively. CONCLUSIONS: Long-term tear volume was not changed after blepharoplasty but was decreased after blepharoptosis surgery, and even more so in cases with an initial high tear volume.

Short-term changes in tear volume after blepharoptosis repair.

PURPOSE: The aim was to evaluate tear volume change by using videomeniscometry before and after blepharoptosis surgery. METHODS: Fifty-nine eyes of 36 patients with blepharoptosis without lacrimal duct obstruction or other eyelid diseases were examined. All the patients underwent levator advancement, and the tear volume was evaluated preoperatively and 1.5 months postoperatively. The margin reflex distance-1 was determined from photographs, and the tear volume was determined by the measurement of tear meniscus radius using videomeniscometry. RESULTS: The mean margin reflex distance-1 was significantly increased after performing a blepharoptosis surgery (0.13 ± 1.13 mm preoperatively vs. 3.18 ± 0.85 mm postoperatively, P < 0.001). The average tear meniscus radius was significantly decreased (0.31 ± 0.16 mm preoperatively vs. 0.23 ± 0.08 mm postoperatively, P < 0.001). A postoperative reduction in the tear meniscus radius was noted in 46 eyes (78%) of 31 patients, and no reduction was noted in 13 eyes (22%) of 12 patients. Preoperative higher tear meniscus radius values were more likely to be decreased (P < 0.01). CONCLUSIONS: The tear volume was decreased after the blepharoptosis surgery was performed, especially for an initially high tear volume.

Small, moderate, and large changes, and the minimum clinically important difference in the University of California, San Diego Shortness of Breath Questionnaire.

Minimum clinically important change of 5 points in the University of California, San Diego Shortness of Breath Questionnaire (SOBQ) is established, but cutoff values between a small, a moderate, and a large change are still unknown. We used the data set of National Emphysema Treatment Trial consisting of severe and very severe chronic obstructive pulmonary disease patients, whose mean age was 64 years. Changes from baseline to post-surgical 6-month follow-up were evaluated. The St. George's Respiratory Questionnaire was used as anchor: |∆SGRQ| < 4, meaningless change; 4 ≤ |∆SGRQ| < 8, small change; 8 ≤ |∆SGRQ| < 13, moderate change; 13 ≤ |∆SGRQ|, large change. We decided the final cutoff values for the SOBQ as medians of the three anchor methods. We also decided the range of cutoff values as the range of three values. In a cohort of surgically treated patients (N = 484), we propose value of 5 (range 5-6), 11 (range 9-15), and 16 (range 14-20) for the cutoff values between a meaningless and a small change (minimum clinically important difference), a small and a moderate change, and a moderate and a large change, respectively. In a cohort of medically treated patients, numbers of patients categorized according to ∆SOBQ scores were similar to those of the patients categorizes according to the ∆SGRQ (N = 480) or ∆Forced expiratory volume in 1 second (N = 425). We propose group-level cutoff values and range between a small, a moderate, and a large changes.

Long-acting beta-agonists reduce mortality of patients with severe and very severe chronic obstructive pulmonary disease: a propensity score matching study.

BACKGROUND: Long-acting beta-agonists were one of the first-choice bronchodilator agents for stable chronic obstructive pulmonary disease. But the impact of long-acting beta-agonists on mortality was not well investigated. METHODS: National Emphysema Treatment Trial provided the data. Severe and very severe stable chronic obstructive pulmonary disease patients who were eligible for volume reduction surgery were recruited at 17 clinical centers in United States during 1988-2002. We used the 6-10 year follow-up data of patients randomized to non-surgery treatment. Hazard ratios for death by long-acting beta-agonists were estimated by three models using Cox proportional hazard analysis and propensity score matching were measured. RESULTS: The pre-matching cohort was comprised of 591 patients (50.6% were administered long-acting beta-agonists. Age: 66.6 ± 5.3 year old. Female: 35.4%. Forced expiratory volume in one second (%predicted): 26.7 ± 7.1%. Mortality during follow-up: 70.2%). Hazard ratio using a multivariate Cox model in the pre-matching cohort was 0.77 (P = 0.010). Propensity score matching was conducted (C-statics: 0.62. No parameter differed between cohorts). The propensity-matched cohort was comprised of 492 patients (50.0% were administered long-acting beta-agonists. Age: 66.8 ± 5.1 year old. Female: 34.8%. Forced expiratory volume in one second (%predicted) 26.5 ± 6.8%. Mortality during follow-up: 69.1%). Hazard ratio using a univariate Cox model in the propensity-matched cohort was 0.77 (P = 0.017). Hazard ratio using a multivariate Cox model in the propensity-matched cohort was 0.76 (P = 0.011). CONCLUSIONS: Long-acting beta-agonists reduce mortality of severe and very severe chronic obstructive pulmonary disease patients.

Preoperative chemotherapy is effective for stage III resectable non--small-cell lung cancer: metaanalysis of 16 trials.

BACKGROUND: The benefit of preoperative chemotherapy for resectable non-small-cell lung cancer is still controversial. PATIENTS AND METHODS: We conducted fixed-model metaanalysis including randomized controlled trials comparing 'preoperative chemotherapy plus surgery' and 'surgery alone' as a primary study with sufficient data to provide a hazard ratio for overall survival. MEDLINE and Cochrane databases were used for the study search. RESULTS: We found 16 studies. Seven included only stage III disease cases, and 9 were conducted without stage limitation. Sixteen trials involving 3728 samples observing 2326 deaths yielded a pooled hazard ratio for overall survival of 0.84 (95% confidence interval [CI], 0.77-0.91; P < .001) with moderate heterogeneity (I(2) = 40%). In sensitivity analysis, strong heterogeneity (I(2) = 69%) was found between the 7 trials covering only stage III disease and 9 trials without stage limitation. The 7 studies evaluating only stage III disease involving 1447 samples and 1068 deaths yielded a pooled hazard ratio of 0.77 (95% CI, 0.68-0.87; P < .001) with nonsignificant low heterogeneity (I(2) = 17%). No publication bias was observed throughout this study. The effect of preoperative chemotherapy differs among stages. The pooled hazard ratio comparing 'preoperative chemotherapy plus surgery' and 'surgery alone' for patients with stage III disease in our study was 0.77, which is slightly better than the pooled hazard ratio of 0.83 in the Lung Adjuvant Cisplatin Evaluation study that compared 'surgery plus postoperative chemotherapy' and 'surgery alone.' CONCLUSION: Preoperative chemotherapy plus surgery for stage III disease is more effective than previously considered.

Mediastinal bronchogenic cyst presenting with pericardial effusion.

We report a case of a 58-year-old woman who had a bronchogenic cyst presenting with pericardial effusion. Initially, the effusion had disappeared prior to resection of the cyst, but it reappeared with additional symptoms. Resection of the bronchogenic cyst resulted in complete resolution of the pericardial effusion and additional symptoms such as progressive dyspnea and chest pain. Application of surgical treatment for asymptomatic cysts in adults remains controversial, but we recommend that mediastinal cysts which present with any symptoms should be resected at the time of their discovery, even though the symptoms disappeared for a while.

Epigenetic silencing of the sulfate transporter gene DTDST induces sialyl Lewisx expression and accelerates proliferation of colon cancer cells.

Colon cancer cells express the carbohydrate determinant sialyl Lewis(x), while they exhibit markedly decreased the expression of its sulfated derivative, sialyl 6-sulfo Lewis(x). In contrast, normal colonic epithelial cells strongly express sialyl 6-sulfo Lewis(x), but they virtually do not express sialyl Lewis(x). Impaired sulfation was therefore suggested to occur during the course of malignant transformation of colonic epithelial cells and was assumed to be responsible for the increased sialyl Lewis(x) expression in cancers. To elucidate the molecular biological background of the impaired sulfation in cancers, we studied the expression levels of mRNA for 6-O-sulfotransferase isoenzymes, PAPS synthases and transporters, and a cell membrane sulfate transporter, DTDST, in cancer tissues. The most striking decrease in cancer cells compared with nonmalignant epithelial cells was noted in the transcription of the DTDST gene (P = 0.0000014; n = 20). Most cultured colon cancer cells had a diminished DTDST transcription, which was restored when cultured with histone deacetylase inhibitors. Suppression of DTDST transcription under the control of a tet-off inducible promoter resulted in increased sialyl Lewis(x) expression and reduced sialyl 6-sulfo Lewis(x) expression. Unexpectedly, the growth rate of the cancer cells was markedly enhanced when transcription of DTDST was suppressed. These results show that the decrease in the transcription of the sulfate transporter gene is the major cause of decreased expression of sialyl 6-sulfo Lewis(x) and increased expression of sialyl Lewis(x) in colon cancers. The results also suggest that the diminished DTDST expression is closely related to enhanced proliferation of cancer cells.

Effect of droplet size on the oxidative stability of soybean oil TAG and fish oil TAG in oil-in-water emulsion.

The effect of droplet size on the oxidative stability of triacylglycerol (TAG)-in water emulsion was examined. Microchannel (MC) emulsification was used to make monodispersed emulsion of soybean oil TAG and fish oil TAG with different droplet size. The main polyunsaturated fatty acids (PUFA) of soybean oil TAG and fish oil TAG were linoleic acid (LA; 53%) and docosahexaenoic acid (DHA; 37.3%), respectively. Oxidation was induced by the addition of 2,2'-azobis(2-amidinopropane)-dihydrochloride (AAPH) or ferrous ion. The oxidative stability was followed by the decrease in the oxygen consumption in the solution, peroxide formation, and decrease in the unoxidized PUFA during oxidation, indicating that the oxidative stability of fish oil TAG increased with decreasing the droplet size, while the reverse effect of the droplet size was observed on the oxidation of soybean oil TAG. The decrease in the droplet size induces the increase in the droplet interface, from which the oxidation proceeds to the oil droplet interior. DHA in fish oil TAG would take highly protective interface against oxidative attack of free radicals and metal ions, whereas LA in soybean oil TAG would be more easily oxidized at the interface because of its less protective conformation. The reverse effect of the droplet size on fish oil TAG and soybean oil TAG could be explained by the different interface conformation of both TAG.

Clinical application of functional glycoproteomics - dissection of glycotopes carried by soluble CD44 variants in sera of patients with cancers.

We provide here an example of clinical application of functional glycoproteomics for cancer diagnosis. Sialyl Lewis a and sialyl Lewis x glycotopes, which are the specific ligands for selectins, and variant forms of CD44, which are the adhesion molecules recognizing hyaluronate, are both implicated in cancer metastasis. The CD44 variants modified by the sialyl Lewis a and sialyl Lewis x glycotopes are expected to have dual functions, serving as ligands for vascular selectins, and simultaneously having binding activity to vascular bed hyaluronate, and are expected to figure heavily in cancer metastasis. We developed a heterogeneous sandwich assay system to detect soluble CD44v specifically modified by the cancer-associated sialyl Lewis a/x glycotopes, using the extracellular domain of CD44v cleaved by the metalloproteinase ADAM10 as standard molecules. We also developed the assay system for CD44v modified by normal epithelial glycotopes including disialyl Lewis a and sialyl 6-sulfo Lewis x. The results indicated that serum levels of soluble CD44v modified by cancer-associated glycotopes were frequently increased in patients with cancers, while those of CD44v modified by the nonmalignant glycotopes tended to be elevated in patients with benign disorders.

Human B-lymphocytes express alpha2-6-sialylated 6-sulfo-N-acetyllactosamine serving as a preferred ligand for CD22/Siglec-2.

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Morphodynamics of ovarian follicles during oogenesis in mice.

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.