Mesdc2 plays a key role in cell-surface expression of Lrp4 and postsynaptic specialization in myotubes

Low-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre- and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation.     

The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis

The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay

The comet assay was performed to elucidate the linearity of calibration curves and detection limits for DNA damage in multiple organs of whole body X-irradiated mice, and rates of reduction in DNA damage by DNA repair during the irradiation period were estimated in the respective organs by comparing the rates of increase in DNA damage at different absorbed dose rates of X-rays. Of the assay parameters, tail length and the percentage DNA in the tail showed a higher sensitivity to DNA damage in most organs than Olive tail moment. Data at the higher absorbed dose rates (2.22 or 1.44 Gy/min) showed good correlations between absorbed doses and these two parameters, with correlation coefficients of more than 0.7 in many organs. However, this assay had difficulty detecting DNA damage at the lower absorption dose rate (0.72 Gy/min). The estimated rates of increase in DNA damage and those of DNA repair during the irradiation period in the respective organs suggested differences in the radiosensitivity of nuclear DNA and DNA repair capacity among organs. Our results indicated that absorbed dose rates of 1.0-1.3 Gy/min or greater were needed to induce detectable DNA damages by the comet assay in many organs.     

Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds.     

Disorders of Orcadian Body Temperature Rhythm in Severely Brain-Damaged Patients

Twenty-four hour patterns of body temperature (BT) were recorded during consecutive 3-10 day spans from 14 severely brain-damaged patients. Seven patients exhibited a normal circadian BT rhythm with an amplitude of more than 1°C and a normal phase position of the minimum BT being observed during the latter half of the nocturnal sleep. One patient with a dispersed type of sleep exhibited an extremely low amplitude of the BT rhythm with mean average 0.69°C. In this patient, an 24-hr observation span was insufficient to detect the existence of a BT rhythm. Two patients manifested disturbance of period. As acrophase of the BT rhythm varied from day to day, the standard deviation (S.D.) of mean acrophase wasextremely large. For these patients the light-dark cycle did not act as an entrainer because both had visual disturbance. A phase advance of the minimum BT was observed in four patients. The minimum BT appeared in the first half of nocturnal sleep. These three disturbances (amplitude, period and phase), were revealed only by longitudinal observation of the BT rhythm, indicating the importance of long-term observations over a sufficient period to make the nature of the rhythm disturbances clear. These disturbances were not related to that of the sleep-wake cycle, as two patients showed normal circadian BT rhythm in spite of their dispersed-type sleep.     

Three cell lines showing androgen-dependent, -independent,and-suppressed phenotypes,established from a single tumor of androgen-dependent shionogi carcinoma 115

Summary We investigated the heterogeneity of cells in terms of androgen responsiveness within a single tumor mass of Shionogi carcinoma SC-115 showing androgen-dependent growth. After cloning of the tumor by the limiting dilution method in the presence of androgen, we isolated 40 clones at random. Twenty-two clones required androgen for growth (androgen-dependent phenotype), 16 did not (androgen-independent phenotype), and the remaining two clones showed growth inhibition when androgen was added (androgen-suppressed phenotype). In addition, 22 androgen-dependent clones showed heterogeneity in growth factor sensitivity in the absence of androgen. All clones were sensitive to both acidic and basic fibroblast growth factor (FGF), 7 of 22 clones were sensitive to epidermal growth factor (EGS) and transforming growth factor (TGF)-α, and 2 of 22 clones were sensitive to TGF-β. This preexisting heterogeneity may be partly responsible for the growth of androgen-dependent tumor under hormone-deprived circumstances. Three typical clones, SC2G, SC1G, and SC4A, were selected from androgen-dependent, -independent, and-suppressed phenotypic groups, respectively. These clones, as well as original solid tumors, were found to produce heparin-binding growth factors of heterogeneous elution positions. The molecular nature of these growth factors is not yet known. Neither anti-basic FGF antibody nor anti-EGF antibody inhibited the cell growth when added in cell culture, suggesting the factors were distinct from basic-FGF and EGF.