Urinary yttrium excretion and effects of yttrium chloride on renal function in rats

Evaluation of yttrium exposure in biological samples has not been fully examined. To evaluate yttrium nephrotoxicity, yttrium chloride was orally administered to male Wistar rats and the urine volume (UV) and N-acetyl-beta-D-glucosaminidase (NAG) and creatinine excretion (Crt) were measured in 24-h urine samples. The urinary yttrium concentration and excretion rate were determined by inductively coupled plasma-argon emission spectrometry (ICP-AES). Large significant decreases of UV (>30%) and Crt (>10%) were observed at yttrium doses of 58.3-116.7 mg per rat, but no significant NAG changes was observed. This response pattern shows that a high yttrium dosage alters glomerular function rather than the proximal convoluted tubules. A urinary yttrium excretion rate of 0.216% and good dose-dependent urinary excretion (r=0.77) were confirmed. These results suggest that urinary yttrium is a suitable indicator of occupational and environmental exposure to this element, an increasingly important health issue because recent technological advances present significant potential risks of exposure to rare earth elements. We propose that the ICP-AES analytical method and animal experimental model described in this study will be a valuable tool for future research on the toxicology of rare earth elements.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

Comparison of stress responses between mental tasks and white noise exposure

The purpose of this study was to compare the cardiovascular responses to different types of mental stress. Ten healthy males performed a mental arithmetic task (MA) on one day and were exposed to white noise (WN, 80dB) on another day. Both the MA and the WN were composed of four 5-min consecutive periods with a 3-min rest between them. On each day, the systolic and diastolic blood pressure (SBP and DBP), mean arterial pressure (MAP), cardiac output (CO), and total peripheral resistance (TPR) were measured continually during the entire experimental period. The changes from the baseline (Delta) in all periods were calculated for both mental stresses. As for the results, the DeltaMAP, DeltaCO, DeltaHR, and DeltaTPR in the MA did not significantly change during the task periods. However, in the WN, the DeltaMAP and DeltaTPR showed significant increases over the time of the consecutive periods. In addition, we discuss the response patterns for the two mental stresses. We examine three hemodynamic reactivity patterns: a cardiac pattern characterized by increased CO and decreased TPR, a mixed pattern characterized by a moderate increase in both CO and TPR, and a vascular pattern characterized by increased TPR and decreased CO. The results show that throughout all task/exposure periods, the response pattern remained the same for six subjects in each stress. Furthermore, of these six subjects, half showed the same response pattern in both the MA and the WN. In conclusion, compared to the MA task, consecutive WN exposure showed an accumulation of stress responses. A change in TPR contributed to a gradual increase in MAP in the WN. It is also possible that among the subjects there were different types of response to the MA and WN.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.