Glycoform analysis of Japanese cedar pollen allergen, Cry j 1

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.     

Resting Heart Rate Variability and the Effects of Biofeedback Intervention in Women with Low-Risk Pregnancy and Prenatal Childbirth Fear

Anxiety about labor in women at the end of pregnancy sometimes reaches levels that are clinically concerning. We investigated whether low-risk pregnant women with childbirth fear during the last trimester demonstrate specific findings with regard to resting heart rate variability (HRV) and examined whether HRV biofeedback can reduce this fear and alter resting HRV. We measured the levels of childbirth fear (Wijma delivery expectancy/experience questionnaire, W-DEQ) and resting HRV indexes in 97 low-risk pregnant women in their 32nd–34th week of gestation and advised women with W-DEQ scores of ≥?66 (n?=?40) to practice HRV biofeedback (StressEraser) at home. We then reassessed these measures 3–4 weeks later in the 36th–37th week of gestation regardless of whether the women practiced the method. We found that childbirth fear had no significant effect on resting HRV indexes when the W-DEQ cutoff was conventionally set at ≥?66. However, women with W-DEQ scores of ≥?90 (n?=?5) had a significantly lower high-frequency power than their counterparts (p?=?0.028). The W-DEQ scores reduced significantly in women who performed HRV biofeedback (n?=?18, p?<?0.001), but there was no change in those who did not perform the method (n?=?20). These findings suggested that very high W-DEQ scores (≥?90), but not the conventional criteria (W-DEQ score?≥?66), of the fear of childbirth were associated with low parasympathetic activity among low-risk pregnant women and that HRV biofeedback intervention can effectively decrease the fear of childbirth in these women.     

Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo–fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo–fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo–fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo–fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo–fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo–fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo–fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.     

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post‐translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin‐like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO‐modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome‐targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome‐targeting is crucially required for the repair of TRF2‐depleted dysfunctional telomeres by 53BP1‐mediated non‐homologous end joining.     

Pentavalent Vanadium at Concentration of the Underground Water Level Enhances the Sweet Taste Sense to Glucose in College Students

Underground water in volcanic areas contains vanadium when the basalt layer exists among igneous rocks. The concentration of vanadium in drinking water sometimes exceeds 0.8 μM in these areas, however, the physiological effects of vanadium, especially non-toxic effects, at concentrations lower than 1 μM are unknown. In the present experiments, we examined the effect of pentavalent vanadium and tetravalent vanadium at 0.8 and 8.0 μM concentrations on the recognition threshold to taste substances in healthy college students. Pentavalent vanadium, ammonium vanadate, lowered the sweet taste threshold to glucose at 0.8 and 8.0 μM as well. Tetravalent vanadium, vanadium sulfate, did not alter the threshold to glucose either at 8.0 μM or at 0.8 μM. Ammonium vanadate also decreased the sweet taste threshold to l-proline at 8.0 μM. Ammonium vanadate did not influence the sour taste threshold to hydrogen chloride. Neither ammonium sulfate nor ammonium bicarbonate altered the sweet taste threshold to glucose. Therefore, the effect of ammonium vanadate on the sweet taste threshold is attained by vanadium but not by ammonium. It was concluded that pentavalent vanadium at 0.8 μM intensifies the sweet taste sense to glucose rather specifically. We have first shown the physiological effect of vanadium at the concentration of the underground water level.