Assessing urine ammonium concentration by urine osmolal gap in chronic kidney disease.

Acidemia is one of the risk factors for end-stage kidney disease and increases the mortality rate of patients with chronic kidney disease (CKD). Although urinary ammonium (U-NH4 + ) is the crucial component of renal acid excretion, U-NH4 + concentration is not routinely measured. To estimate U-NH4 + , urine osmolal gap (UOG = urine osmolality - [2(Na+ + K+ ) + urea + glucose]) is calculated and the formula (U-NH4 +  = UOG/2) has traditionally been used. However, the usefulness of this formula is controversial in CKD patients. We assessed the relationship between U-NH4 + and UOG in patients with CKD. Blood and spot urine samples were collected in 36 patients who had non-dialysis-dependent CKD. The mean ± SD age of patients was 72.0 ± 14.8 years, and the mean ± SD serum creatinine and U-NH4 + were 2.7 ± 2.3 mg/dl and 9.3 ± 9.2 mmol/L, respectively. A significant relationship was found between UOG/2 and U-NH4 + (r = .925, p < .0001). U-NH4 + estimated using the UOG was on average higher by 4.7 mmol/L than the measured one. Our results suggested that UOG could be a useful tool in clinical settings, especially in patients with moderate to severe CKD.

Serial measurement of electrolyte and citrate concentrations in blood-primed continuous hemodialysis circuits during closed-circuit dialysis.

BACKGROUND: For continuous renal replacement therapy in small infants, due to the large extracorporeal volume involved, blood priming can be necessary to prevent hypotension and hemodilution. Because packed red blood cells (RBCs) have high levels of potassium and citrate, closed-circuit dialysis is often performed. We assessed the metrics of closed-circuit dialysis and serial citrate concentration changes. METHODS: We performed dialysis of closed circuits primed with expired human packed RBC solution and 5% albumin. Blood and dialysate flow rates were 70 and 33.3 mL/min, respectively. The extracorporeal volume was 70 mL. We measured pH, electrolytes, and citrate in the closed circuit every 3 min for 15 min. We also assessed the adequacy of closed-circuit dialysis using the formula: [dialysate flow rate (mL/min) × time of dialysis (min)]/extracorporeal volume (mL) and we assessed the correlation between citrate and ionized calcium concentrations. RESULTS: To reach normal concentrations of sodium, potassium, and chloride, 2.4 times as much dialysate fluid as extracorporeal volume was needed. In contrast, for ionized calcium, bicarbonate, and citrate, 3.8 times as much dialysate fluid as extracorporeal volume was required. By simple linear regression analysis, the concentration of citrate was significantly correlated with that of ionized calcium. CONCLUSIONS: For closed-circuit dialysis using an RBC solution, the formula [dialysate flow rate (mL/min) × time of dialysis (min)]/extracorporeal volume (mL) would be a better parameter to estimate efficacy, compared with other metrics. Additionally, the citrate concentration can be readily estimated from the ionized calcium concentration during closed-circuit dialysis.

Constitutional abnormalities of IDH1 combined with secondary mutations predispose a patient with Maffucci syndrome to acute lymphoblastic leukemia.

Maffucci syndrome is a nonhereditary disorder caused by somatic mosaic isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) mutations and is characterized by multiple enchondromas along with hemangiomas. Malignant transformation of enchondromas to chondrosarcomas and secondary neoplasms, such as brain tumors or acute myeloid leukemia, are serious complications. A 15-year-old female with Maffucci syndrome developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A somatic mutation in IDH1 was detected in hemangioma and leukemic cells. KRAS mutation and deletion of IKZF1 were detected in leukemic cells. Patients with Maffucci syndrome may, therefore, be at risk of BCP-ALL associated with secondary genetic events that affect lymphocyte differentiation.

A breast milk production model for achieving exclusive breastfeeding in low-risk primiparas who had vaginal births at 4 months postpartum: A prospective cohort study.

BACKGROUND: This study aimed to investigate the thresholds of milk production for predicting the achievement of exclusive breastfeeding (EBF) at 4 months postpartum. METHODS: The inclusion criteria were singleton, low-risk primiparas, non-cesarean section, and healthy mothers and infants. Temporary feeding of artificial milk in the past was included. Women who could be judged to have intentionally used artificial milk were excluded from the analysis. Measurements of the 24-h milk production by test weights were obtained at early postpartum (days 3-4) and at 1 month postpartum. To predict the achievement of EBF at 4 months postpartum, the area under the receiver operating characteristic (ROC) curve was calculated for early postpartum and 1 month milk production. RESULTS: There were 80 eligible participants analyzed. ROC analysis of milk production for achieving EBF at 4 months revealed an area under the curve (AUC) of 0.736 (95% CI: [0.625, 0.848]) for early postpartum and an AUC of 0.854 (95% CI: [0.772, 0.936]) for 1 month postpartum. The proposed thresholds are 102 g/day (higher sensitivity) and 161 g/day (higher specificity) at early postpartum, and 527 g/day (higher sensitivity) at 1 month postpartum. CONCLUSIONS: In low-risk primiparas, the milk production models at early postpartum and 1 month postpartum showed a high accuracy for predicting the achievement of EBF at 4 months postpartum. The present study indicates that even when formula milk is temporarily used, it is still possible to reach the goal of achieving EBF.

A longitudinal study of the association of epidural anesthesia and low-dose synthetic oxytocin regimens with breast milk supply and breastfeeding rates.

Breastfeeding is known to improve maternal and child health. However, epidural anesthesia (EDA) and synthetic oxytocin (synOT) are suggested to have negative effects on breastfeeding. In this study, we aimed to determine the effects of intrapartum synOT and EDA on breast milk supply, breastfeeding rates, and maternal salivary oxytocin levels. Women were recruited during pregnancy or after birth at a single hospital. Data were collected at 3 days postpartum (T1), 1 month postpartum (T2), and 4 months postpartum (T3) on 83 low-risk primiparous women who planned to breastfeed for at least 12 weeks postpartum to avoid dropouts from early discontinuance of breastfeeding. Women with cesarean section, twin pregnancy, premature neonates, and an Apgar score of < 7 at 5 min were excluded. Participants recorded their 24-h milk supply by test weights at 3 days and 1 month postpartum. Additionally, they filled out questionnaires assessing their breastfeeding level and lactogenesis stage II initiation. Salivary oxytocin levels were obtained at 3 days postpartum. Women who delivered using EDA had lower salivary oxytocin levels (P = .055, d = .442), breast milk supply in early postpartum (P = .025, d = .520) and at 1 month postpartum (P = .036, d = .483), and breastfeeding rates at 4 months postpartum (P = .037, V = .236) than women who did not deliver using EDA. There was no association between breastfeeding and the use of intrapartum synOT. In conclusion, this study showed that women who delivered using EDA had lower breast milk supply in the early postpartum period and breastfeeding rates at 4 months postpartum. It also revealed that using synOT at low doses during labor did not affect breastfeeding. Thus, women who deliver using EDA need support for increased breast milk supply in the early postpartum period.Trial registration: UMIN000037783 (Clinical Trials Registry of University Hospital Information Network).

Determination of optimal 25-hydroxyvitamin D cutoff values for the evaluation of restless legs syndrome among pregnant women.

STUDY OBJECTIVES: Vitamin D deficiency is associated with restless legs syndrome (RLS). However, a cutoff value for serum 25-hydroxyvitamin D (25[OH]D) level associated with RLS has yet to be clearly determined. We evaluated the association between 25(OH)D and RLS in pregnant women. METHODS: Data from 203 pregnant women were evaluated using blood samples taken in the third trimester. Liquid chromatography-tandem mass spectrometry and ligand binding assays were used to measure 25(OH)D. RLS was diagnosed based on International Classification of Sleep Disorders, third edition, criteria. The cutoff value for serum 25(OH)D associated with RLS was explored using receiver operating characteristic (ROC) curve and classification and regression tree (CART) analyses. RESULTS: The results of liquid chromatography-tandem mass spectrometry (x) and ligand binding assays (y) for serum 25(OH)D in the RLS (n = 35, 17.2%) and non-RLS (n = 168) groups showed a relationship of y = -2.65 + 0.08x . The RLS group showed lower serum 25(OH)D and folate levels. ROC curve and CART analyses revealed cutoff values of 10-12.7 ng/mL and 6.6-7.2 ng/mL for 25(OH)D and folate, respectively. Of the 5 women with RLS symptoms persisting at a moderate-to-severe level after delivery, 4 had 25(OH)D levels < 10 ng/mL and all had folate levels < 6 ng/mL. CONCLUSIONS: Vitamin D and folate deficiency were associated with RLS in pregnant women and may be associated with persistent moderate-to-severe postpartum RLS symptomatology; it is essential to examine associations with RLS while accounting for measurement methods and assay systems. CITATION: Miyazaki A, Takahashi M, Shuo T, Eto H, Kondo H. Determination of optimal 25-hydroxyvitamin D cutoff values for the evaluation of restless legs syndrome among pregnant women. J Clin Sleep Med. 2023;19(1):73-83.

Changes in Salivary Oxytocin Level of Term Pregnant Women after Aromatherapy Footbath for Spontaneous Labor Onset: A Non-Randomized Experimental Study.

BACKGROUND: Aromatherapy is usually used to stimulate labor. However, its specific physiological effects have been scarcely examined. We evaluated whether an aromatherapy footbath increases oxytocin levels in term pregnant women. METHODS: In this quasi-experimental study, low-risk term pregnant women in Japan underwent aromatherapy using a footbath (1) infused with clary sage and lavender essential oils, (2) infused with jasmine oil, or (3) with no infused oils (control group). The primary outcome was the salivary oxytocin level. The secondary outcomes were uterine contractions and cortisol levels. RESULTS: In the clary sage and lavender group (n = 28), the oxytocin level increased significantly after the footbath (p = 0.035). The jasmine group (n = 27) and control group (n = 27) exhibited trends toward a respective increase and decrease in the oxytocin level; however, the changes in the oxytocin levels between the clary sage and lavender group and the control group showed no significance difference. There were no significant differences in the changes in the uterine contractions and cortisol levels between the experiment and control groups. CONCLUSIONS: The changes in the oxytocin levels in the clary sage and lavender group did not differ significantly with those in the control group, possibly because of the small sample size. Further studies are required to examine the effects of repeated aromatherapy footbaths to stimulate labor.

ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain.

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, ß-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to ß-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

Effects of epidural anesthesia on postpartum maternity blues and fatigue and its relation to changes in oxytocin.

AIMS: This study aimed to clarify (a) the association of the delivery mode (i.e., epidural anesthesia or no epidural) with women's changes in longitudinal salivary oxytocin (OT) levels from late pregnancy to early postpartum, and (b) the association of these changes with postpartum maternity blues (MB) and fatigue. METHODS: We used a longitudinal observational design. We performed measurements of the saliva samples at four measurement points: (a) 36-37 gestational weeks, (b) 38-39 gestational weeks, (c) 1-2 days after birth, and (d) 4-5 days after birth. The inclusion criterion was low-risk primiparous women who were planning to have vaginal delivery. We used Stein's MB scale to evaluate postpartum data and a visual analog scale to examine "postpartum fatigue". We determined the association between the MB/fatigue scores and the changes in the OT levels by Spearman's rank correlation coefficient. RESULTS: There were 29 women who delivered with epidural anesthesia and 29 women who did not. A significantly higher mean salivary OT level was observed in the women with epidural anesthesia at 1-2 days after birth than at 36-37 gestational weeks. The mean OT level at 4-5 days postpartum was significantly lower than that at 1-2 days postpartum. The correlation of the MB score with the salivary OT level at 4-5 days postpartum was ρ = -0.33, p = .01. CONCLUSIONS: Early postpartum OT level decreased with epidural anesthesia and showed a negative correlation with postpartum MB and fatigue. Healthcare providers should recognize that women who had epidural anesthesia need specific supportive care.

Changes in salivary oxytocin levels and bonding disorder in women from late pregnancy to early postpartum: A pilot study.

OBJECTIVES: This pilot study aimed to 1) follow the longitudinal changes in the salivary oxytocin level of pregnant women from late pregnancy to early postpartum, 2) examine the factors related to these changes, and 3) clarify the association of these changes with mother-infant bonding. METHODS: This study used a longitudinal observational design and questionnaires to obtain objective and subjective data. For oxytocin evaluation, saliva samples were collected and their oxytocin levels were measured at 4-time points [i.e., 1) 36-37 gestation weeks, 2) 38-39 gestation weeks, 3) 1-2 days postpartum, 4) 4-5 days postpartum]. The oxytocin level was assayed in duplicates by enzyme-linked immunosorbent assay. Baseline data were evaluated using the Parental Bonding Instrument (25 items), State Trait Anxiety Inventory (20 items), and Center for Epidemiologic Studies Depression Scale. Postpartum data were evaluated using the Mother to Infant Bonding Scale Japanese Version (10 items), Maternity Blues Scale (13 items), and 'Fatigue after Childbirth' using the Visual Analogue Scale (VAS: 0-100 mm). RESULTS: The participants were 13 primiparas with a mean age of 33 years. They had no depression or anxiety at the baseline. Their mean salivary oxytocin levels significantly increased from late pregnancy (36-39 gestation weeks) up to 1 day postpartum and then decreased until 5 days postpartum. There was a negligible correlation between the bonding disorder and the salivary oxytocin level on the 5th day after childbirth. A moderate correlation was observed between the maternity blues score and the salivary oxytocin level. There was a significant negative correlation between the postpartum fatigue and the salivary oxytocin level 1 day and 5 days after childbirth. CONCLUSION: The mean salivary oxytocin levels significantly increased from the baseline up to 1 day postpartum and then decreased until 5 days postpartum. The salivary oxytocin level was moderately associated with maternity blues and significantly with postpartum fatigue.

Effects of breast stimulation for spontaneous onset of labor on salivary oxytocin levels in low-risk pregnant women: A feasibility study.

OBJECTIVES: This preliminary study aimed to 1) determine changes in the salivary oxytocin (OT) level during breast stimulation for promoting the spontaneous onset of labor in low-risk term pregnancies, and 2) clarify the feasibility of the breast stimulation intervention protocol in terms of practicality and acceptability. METHODS: We used a single arm trial design. Sixteen low-risk pregnant women between 38 and 40 weeks of gestation with cephalic presentation participated. They performed breast stimulation for 3 days with an attendant midwife in a single maternity hospital. Each breast was stimulated for 15 minutes for a total of 1 hour per day. Saliva was collected 10 minutes before the intervention and 15, 30, 60, 75, and 90 minutes after the intervention, yielding 18 samples per woman. RESULTS: Among a total of 282 saliva samples from the 16 participants, OT level was measured in 142 samples (missing rate: 49.6%). The median OT level showed the highest values on day 3 of the breast stimulation, with a marked increase 30 min after the intervention. In the mixed models after multiple imputation for missing data, the OT level on the first day of intervention was significantly lower than that on the third day of intervention. Fatigue from breast stimulation decreased on subsequent days, and most of the women (75%) felt no discomfort with the protocol. Uterine hyperstimulation was not observed. CONCLUSION: Following a 3-day breast stimulation protocol for spontaneous onset of labor, the mean OT level showed the highest values on day 3. The breast stimulation intervention protocol showed good feasibility in terms of practicality and acceptability among the pregnant women. Additional large-scale studies are warranted to confirm the protocol's effectiveness.

Changes in salivary oxytocin after inhalation of clary sage essential oil scent in term-pregnant women: a feasibility pilot study.

OBJECTIVES: This pilot study using a quasi-experimental design was conducted to evaluate the feasibility (i.e., limited efficacy, practicality, and acceptability) of our intervention protocol involving inhalation of the scent of clary sage essential oil by pregnant women and measurement of their preinhalation and postinhalation oxytocin levels. RESULTS: Participants were women of singleton pregnancies between 38 and 40 gestation weeks (N = 11). The experiment group (n = 5) inhaled the scent of clary sage essential oil diluted 50-fold with 10 mL of odorless propylene glycol for 20 min. Regarding limited efficacy, the oxytocin level 15 min postinhalation increased in 3 women and was unmeasurable in 2. The control group (n = 6) inhaled similarly without the 50-fold dilution of clary sage essential oil. Their oxytocin level increased in 2 women, decreased in 2, and was unmeasurable in 2. Uterine contraction was not observed in both groups. Regarding practicality, 3 of the 11 women could not collect sufficient saliva. The cortisol level decreased in both groups postinhalation. The protocol had no negative effects. Regarding acceptability, burden of the protocol was not observed. Trial registration The Clinical Trials Registry of University Hospital Medical Information Network in Japan-UMIN000017830. Registered:  June 8, 2015.

Sublethal Photothermal Stimulation with a Micropulse Laser Induces Heat Shock Protein Expression in ARPE-19 Cells.

Purpose/Aim of the Study. Subthreshold micropulse diode laser photocoagulation is an effective treatment for macular edema. The molecular mechanisms underlying treatment success are poorly understood. Therefore, we investigated the effects of sublethal laser energy doses on a single layer of densely cultured ARPE-19 cells as a model of the human retinal pigment epithelium (RPE). Materials and Methods. A single layer of densely cultured human ARPE-19 cells was perpendicularly irradiated with a micropulse diode laser. Nonirradiated cells served as controls. Sublethal laser energy was applied to form a photocoagulation-like area in the cultured cell layers. Hsp70 expression was evaluated using quantitative polymerase chain reaction and immunocytochemistry. Results. Photocoagulation-like areas were successfully created in cultured ARPE-19 cell layers using sublethal laser energy with our laser irradiation system. Hsp70 mRNA expression in cell layers was induced within 30 min of laser irradiation, peaking at 3 h after irradiation. This increase was dependent on the number of laser pulses. Hsp70 upregulation was not observed in untreated cell layers. Immunostaining indicated that Hsp70 expression occurred concentrically around laser irradiation sites and persisted for 24 h following irradiation. Conclusion. Sublethal photothermal stimulation with a micropulse laser may facilitate Hsp70 expression in the RPE without inducing cellular damage.

Detection of the heterogeneous O-glycosylation profile of MT1-MMP expressed in cancer cells by a simple MALDI-MS method.

BACKGROUND: Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently. METHODS/PRINCIPAL FINDINGS: A new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation. CONCLUSIONS/SIGNIFICANCE: We demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis.

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.

Testosterone content of developing eggs and sex reversal in the medaka (Oryzias latipes).

To understand the effect of testosterone on sex differentiation, the quantities of testosterone (T) and estradiol-17beta (E2) in developing eggs of medaka (Oryzias latipes) were measured by radioimmunoassay, and the influence on sex differentiation of treating embryos with exogenous androgens was also examined. Endogenous T of eggs dispersed into the environmental water at spawning, and precipitously declined to a minimum level during incubation for 2 days post-fertilization (dpf). It did not significantly increase during development. The E2 content of fertilized eggs increased when eggs were incubated in medium containing exogenous T at the concentrations of 100 and 500 ng/ml, but not in low concentrations of 10 ng/ml or less. The presence of 500 ng/ml 17alpha-methyltestosterone (MT) in the incubation medium also induced an increase in the E2 content of embryos. Exposure of embryos to exogenous 1 ng/ml T that corresponded with the level of T in eggs shortly after fertilization was enough to induce sex reversal of genotypic females to functional males. The co-existence of T and aromatase inhibitor in incubation medium inhibited not only the T-induced increase in the embryonic E2 content, but also the estrogenic effect of T in causing the paradoxical sex reversal from genotypic males to phenotypic females. However, treatment of embryos with the non-aromatizable androgen, 17alpha-methyldihydrotestosterone, induced no detectable increase in the E2 content of embryos, but still brought about sex reversal of genotypic males into females. This contradictory result suggests that the conversion of androgens to E2 may not always be the cause for induction of paradoxical sex reversal by T treatment. Consequently, these results on sex reversal induced by treatment of embryos with exogenous androgens suggest that endogenous T of developing medaka embryos may not act as the natural andro-inducer, and that genotypic sex can be modified by exogenous sex steroids at early developmental stages long before gonadal differentiation in the medaka.

Identification and functions of chondroitin sulfate in the milieu of neural stem cells.

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.

Expression and identification of a new splice variant of neuroglycan C, a transmembrane chondroitin sulfate proteoglycan, in the human brain.

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan with an EGF module. We studied the expression of NGC in the human brain, mainly in the hippocampus, and confirmed some observations by conducting experiments using rat brain. In humans, NGC mRNA was expressed exclusively in the brain, especially in the immature brain. The telencephalon, including the hippocampus and neocortex, showed strong mRNA expression. NGC was immunolocalized to neuropils in the hippocampus and neocortex of the adult rat. RT-PCR experiments showed that four splice variants (NGC-I, -II, -III, and -IV) were expressed in the adult human hippocampus. By Western blotting, the expression as proteins of all splice variants except NGC-II was confirmed in the adult rat hippocampus. NGC-IV, which was first found in the present study, had the shortest cytoplasmic domain among the four variants. NGC-IV mRNA was expressed by neurons, but not by astrocytes, in culture prepared from the fetal rat hippocampus, suggesting that NGC-IV plays a role specific to neurons. In addition, the human NGC gene, which is registered as CSPG5, comprised six exons and was approximately 19 kb in size. In exon 2, a single nucleotide polymorphism resulting in Val188Gly in the NGC ectodomain was observed.

Estradiol-17beta content in developing eggs and induced sex reversal of the medaka (Oryzias latipes).

To clarify the effect of exogenous estradiol-17beta (E2) on sex differentiation, the E2 content of developing eggs of Oryzias latipes was measured by radioimmunoassay. Endogenous E2 was present in lower concentrations in ovulated, mature eggs in the ovarian cavity than in intrafollicular oocytes. The E2 content of eggs precipitously declined to a minimum level by 2 days post-fertilization. The E2 content of eggs was affected by 24 hr of incubation in medium containing exogenous E2 at concentrations above 10 ng/ml. Short (24 hr) exposure of fertilized eggs in the early developmental stage to exogenous E2 at concentrations of 10 ng/ml induced sex reversal of some genotypic males to functional females. However, endogenous E2 levels in fertilized eggs might not influence sexual differentiation in embryogenesis. The present results suggest the possibility that concentrations of exogenous E2 higher than that of endogenous E2 triggers a priming step in the cascade of sex differentiation toward the female, and this effect is maintained.