Comparison between effects of dantrolene and nifedipine on lipopolysaccharide-induced endotoxemia in the anesthetized rats

Intracellular calcium is an important mediator for regulating the cellular response in endotoxemia. In this study, we investigated the effects of dantrolene and nifedipine, two agents of reducing intracellular calcium levels, on bacterial endotoxin (lipopolysaccharide, LPS; 10 mg/kg i.v.)-induced production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) as well as hemodynamic changes in the anesthetized rat. Injection of LPS (i) induced biphasic changes of blood glucose and rectal temperature: an initial increased phase (<180 min after injection of LPS) followed by a decreased phase (at 240 or 360 min), (ii) caused a significant fall in mean arterial blood pressure from 119+/-3 mmHg (at time 0) to 73+/-67 mmHg (at 360 min) with a concomitant increase of heart rate, (iii) resulted in a substantial hyporeactivity to norepinephrine (NE) (1 microg/kg i.v.), (iv) increased plasma nitrate (an indicator of NO formation) in a time-dependent manner, and (v) induced bell-shape changes in plasma TNF-alpha levels which reached a peak at 60 min. Pretreatment of animals with dantrolene (1 mg/kg i.v. at 20 min prior to LPS) or nifedipine (20 microg/kg i.v. infusion for 20 min at 20 min prior to LPS) not only attenuated the delayed circulatory failure (e.g. delayed hypotension and vascular hyporeactivity to NE), but also prevented the overproduction of NO caused by LPS in the rat. However, the prevention of NO overproduction by dantrolene, but not by nifedipine, was associated with an inhibition of TNF-alpha production elicited by LPS. Thus, both dantrolene and nifedipine have beneficial hemodynamic effects, although through different mechanisms, in animals with endotoxic shock.     

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto-temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3-relative to 4-repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.     

Natural antimicrobial agent (reuterin) produced by lactobacillus reuteri for sanitization of biological tissues inoculated with pseudomonas aeruginosa

The study was done to evaluate the efficacy of using reuterin produced by Lactobacillus reuteri to sanitize biological tissues. The microorganism tested in the study was Pseudomonas aeruginosa, a common cause of nosocomial biomaterial-related infections. The inhibitory effect of reuterin on P. aeruginosa for an inoculated tissue was investigated at different conditions of concentration, temperature, and pH. Additionally, the cellular compatibility of the reuterin-sanitized tissue was evaluated. Glutaraldehyde was employed as a control. It was noted that the minimum inhibitory concentration (MIC, 33.0 +/- 2.9 ppm) and minimum bactericidal concentration (MBC, 50.0 +/- 0.0 ppm) values of reuterin for P. aeruginosa were significantly lower than their glutaraldehyde counterparts (MIC, 130.0 +/- 8.2 ppm and MBC, 180.0 +/- 18.3 ppm). This indicated that reuterin was more efficient than glutaraldehyde as an antimicrobial agent. The addition of reuterin on the inoculated tissue led to a reduced viability of P. aeruginosa. The reduction in the P. aeruginosa culture was more pronounced with increasing the concentration of reuterin (0-100 ppm). At increasing temperature (25-45 degrees C), there was an increasing effect of reuterin on its sanitization activity. However, it should be pointed out that the growth of P. aeruginosa in the nutrient broth was also significantly affected by temperature. The sanitization activity of reuterin was more evident with increasing the pH level (pH 6.5-8.5). The cytotoxicity of reuterin was significantly lower than that of glutaraldehyde. Additionally, the cellular compatibility of the reuterin-sanitized tissue was superior to its glutaraldehyde-sanitized counterpart.     

Schizosaccharomyces pombe Int6 and Ras homologs regulate cell division and mitotic fidelity via the proteasome

Yin6 is a yeast homolog of Int6, which is implicated in tumorigenesis. We show that Yin6 binds to and regulates proteasome activity. Overexpression of Yin6 strengthens proteasome function while inactivation weakens and causes the accumulation of polyubiquitinated proteins including securin/Cut2 and cyclin/Cdc13. Yin6 regulates the proteasome by preferentially interacting with Rpn5, a conserved proteasome subunit, and affecting its localization/assembly. We showed previously that Yin6 cooperates with Ras1 to mediate chromosome segregation; here, we demonstrate that Ras1 similarly regulates the proteasome via Rpn5. In yeast, human Int6 binds Rpn5 and regulates its localization. We propose that human Int6, either alone or cooperatively with Ras, influences proteasome activities via Rpn5. Inactivating Int6 can lead to accumulation of mitotic regulators affecting cell division and mitotic fidelity.     

Rpn5 is a conserved proteasome subunit and required for proper proteasome localization and assembly

Proper function of the 26 S proteasome requires assembly of the regulatory complex, which is composed of the lid and base subcomplexes. We characterized Rpn5, a lid subunit, in fission yeast. We show that Rpn5 associates with the proteasome rpn5. Deletion (rpn5Delta) exacerbates the growth defects in proteasome mutants, leading to mitotic abnormalities, which correlate with accumulation of polyubiquitinated proteins, such as Cut2/securin. Rpn5 expression is tightly controlled; both overexpression and deletion of rpn5 impair proteasome functions. The proteasome is assembled around the inner nuclear membrane in wild-type cells; however, in rpn5Delta cells, proteasome subunits are improperly assembled and/or localized. In the lid mutants, Rpn5 is mislocalized in the cytosol, while in the base mutants, Rpn5 can enter the nucleus, but is left in the nucleoplasm, and not assembled into the nuclear membrane. These results suggest that Rpn5 is a dosage-dependent proteasome regulator and plays a role in mediating proper proteasome assembly. Moreover, the Rpn5 assembly may be a cooperative process that involves at least two steps: 1) nuclear import and 2) subsequent assembly into the nuclear membrane. The former step requires other components of the lid, while the latter requires the base. Human Rpn5 rescues the phenotypes associated with rpn5Delta and is incorporated into the yeast proteasome, suggesting that Rpn5 functions are highly conserved.     

Analysis of secretory immunoglobulin A in human saliva by laser-induced fluorescence capillary electrophoresis

The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He-Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA-Cy5 was mixed with antibody or anti-sIgA-Cy5 mixed with sIgA to form Ag-Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag-Ab complex peak area.     

The effect of prior prolonged low frequency stimulation on the further synaptic plasticity at hippocampal CA1 synapses

The objective of this study is to determine the role of prior prolonged low frequency stimulation (900 pulses at 1 Hz) on the further induced long-term potentiation (LTP) and depression (LTD) of synaptic activity in the rat hippocampal CA1 area. Hippocampal slices and standard extracellular field potential recording techniques were employed. LTP and LTD were induced using stimulation at 5 Hz (900 pulses) paired with or without simultaneous application of 1 microM isoproterenol respectively, at either normal CA1 synapses or CA1 synapses that were pre-conditioned with prolonged low frequency stimulation at 1 Hz. LTD could be successfully induced upon 900 pulses of stimulation given at 5 Hz at normal synapses (82.1 +/- 2.9%; n = 5); it was, however, reduced to 96.5 +/- 4.7% (n = 6) at the preconditioned synapses. When paired with application of isoproterenol, 900 pulses of stimulation given at 5 Hz produced LTP (139.9 +/- 9.6%, n = 5) at normal synapses. The magnitude of LTP is decreased to (130 +/- 13.2%) (n = 6) at pre-conditioned synapses, though the difference is not significant. These results suggest that at a given CA1 synapses the expression of LTP and LTD is dependent on their history of use.