Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.     

Refolding of thioredoxin reductase assisted by groEL and PDI

Thioredoxin reductase was unfolded in 2 M guanidine hydrochloride as revealed by fluorescence and CD spectroscopy. Spontaneous refolding of denatured species resulted in low recovery of 10% catalytic activity after 4 h incubation at 25 degrees C. Addition of groEL or protein disulfide isomerase to the renaturation buffer accelerated the rate of recovery of catalytic activity to a level of 35 and 15%, respectively. Fluorescence spectroscopy has been used to investigate the interaction of groEL and protein disulfide isomerase with denatured thioredoxin reductase tagged with a fluorescent probe. The fluorescence emitted by the denatured protein was quenched upon binding to either groEL or protein disulfide isomerase. It is suggested that encapsulation of the protein substrate by the chaperone plays an important role in the process of folding by facilitating the formation of correctly folded species.     

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.     

Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and ²H₂O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to ²H₂O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from ²H₂O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes     

Use of a permanent magnetic field to inhibit the development of canine osteoarthritis

This study was designed to determine the potential of a permanent magnetic field to inhibit the progression of osteoarthritis (OA) in a canine model. The magnetic field was created by 72 domino-sized ceramic magnets with surface field strength of 1100 G (0.11 T). The magnetic field strength at the surface of the mattress was 450-500 G (45-50 mT) and was equally distributed over the mattress surface. Eighteen animals had closed resection of their right stifle anterior cruciate ligament. Their kennel floors were covered in one of three ways: no floor mattress (OA) (N = 6); a floor mattress with domino-sized ceramic pieces placed between two layers of foam (sham control OA-MAT) (N = 6); or a floor mattress with domino-sized ceramic permanent magnets placed between two layers of foam (OA-MAT-MAG) (N = 6). Animals were kept in their cages except for 4 h of exercise each day. The left stifle of six animals served as the normal control. The stifle joints were examined at 12 weeks for synovial effusion, gross anatomic appearance, microscopic anatomic appearance (Mankin score), and metalloproteinase (MMP)-1 and -3. Macroscopically, the OA-MAT-MAG group appeared to have less synovitis, less synovial effusion, less disruption of the cartilage surface, and less cartilage ulceration than did the OA group or the control mattress group. The mean Mankin score for the OA-MAT-MAG group was less than that for the OA group (4.2 +/- 0.8 vs. 6.7 +/- 0.3; P <.05) and the control mattress group (4.2 +/- 0.8 vs. 5.2 +/- 0.8; P >.05), but greater than that for the normal left group (4.2 +/- 0.8 vs. 1.0 +/- 0.4; P <.05). These scores show a trend of improvement for OA-MAT-MAG group but the difference with the sham control OA-MAT group was not statistically significant. In immunohistochemical studies, the OA-MAT-MAG group cartilage was stained less heavily for MMP-1 and MMP-3 than were the OA group cartilage and the control mattress group cartilage, but did not differ significantly in MMP-1 and MMP-3 from the normal left group cartilage. The OA-MAT-MAG group did not differ from the normal left group in MMP-3 as determined by Western blot analysis. The study suggests that OA of the medial femoral condyle developed in a canine model exposed to a magnetic field may be inhibited beyond the benefit provided by mattress. Further studies are needed to delineate more precisely the effect of the magnetic field in reducing the severity of OA.     

A common open representation of mass spectrometry data and its application to proteomics research

A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.     

Multiplicity of Ca2+ messengers and Ca2+ stores: a perspective from cyclic ADP-ribose and NAADP

It is generally believed that multiple Ca2+ stores are present in cells, a notion that has now been made substantive by the discovery of multiple Ca2+ mobilizing messengers. Cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP) are two such messengers that are derived from NAD and NADP, respectively. A wide variety of cells, from plants to mammals, including human, have been shown to be responsive to these two novel Ca2+ messengers. Not only are their structures and mechanisms of action different, their targeted Ca2+ stores are also distinct and separable. This article explores the implications of the multiplicity of Ca2+ stores in cellular signaling. Special emphasis will be put on the recent progress in the understanding of the physiological functions of NAADP.