End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.     

Evaluation of Vitek GPS-SA card for testing of oxacillin against borderline-susceptible staphylococci that lack mec

Fifty-one Staphylococcus aureus strains lacking mec for which oxacillin MICs were 1 to 8 micrograms/ml were tested against oxacillin and the combination of oxacillin and clavulanic acid with the Vitek GPS-SA card, the reference broth microdilution method, and the oxacillin agar screen plate. Of the 51 strains, 44 (86%) did not grow on the oxacillin agar screen plate, broth microdilution MICs were 1 to 2 micrograms/ml, and GPS-SA card MICs were < or = 2 micrograms/ml, with the exception of 3 strains that failed to grow in the card on repeated attempts. Another seven strains did grow on the oxacillin agar screen plate. For four of the latter group of strains, oxacillin broth microdilution MICs were > 4 micrograms/ml and GPS-SA card MICs were > or = 4 micrograms/ml; for the other 3 strains, corresponding MICs were 4 and < or = 2 micrograms/ml, respectively. The GPS-SA card classified 86% of strains as oxacillin susceptible.     

The maternal nudel protein of Drosophila has two distinct roles important for embryogenesis

The nudel gene is maternally required to define dorsoventral polarity of the Drosophila embryo. It encodes an unusual mosaic protein with a protease domain that may trigger the protease cascade required for ventral development. We describe phenotypic and molecular analyses of nudel mutations that provide further insight into nudel protein function. Surprisingly, nudel mutations primarily cause either dorsalized embryos in which dorsal cell fates are expanded over ventral and lateral cell fates or fragile eggs that fail to develop beyond early embryonic stages. The nudel protein is therefore required not only for embryonic dorsoventral polarity but also for structural integrity of the egg. Complementation and antagonistic interactions between nudel alleles suggest that the nudel protein is functionally modular and that protein-protein interactions are important for nudel protein function. Three nudel mutations that produce dorsalized embryos map to the protease domain of nudel, suggesting that this domain is specifically required for defining embryonic dorsoventral polarity. Finally, certain combinations of nudel alleles simultaneously produce completely dorsalized and normal embryos yet very few embryos of intermediate mutant phenotypes. The unusual biphasic distribution of phenotypes may indicate that nudel activity above a threshold is required to generate embryonic dorsoventral polarity.     

Hypoxia-ischemia induces a rapid elevation of ubiquitin conjugate levels and ubiquitin immunoreactivity in the immature rat brain

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia-ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.     

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.     

Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells

Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin- Hoechst(low)/Rhodamine(low) (Lin- Ho(low)/Rho(low)) and 10% to 15% of Lin-/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin- Ho(low)/Rho(low) cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0/G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin- Rho(low)/Ho(low) cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin- Ho(low)/Rho(low) cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin-/Sca+ population of bone marrow cells. Stem cell factor (SCF) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin-/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin-/Sca+ cells at G1/S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1. For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells.     

Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma

A 56-year-old female with rheumatoid arthritis was admitted because of bilateral hip pain. In a few months of her hospitalization, a relatively abrupt renal dysfunction was emerged besides complement breakdown, and renal biopsy revealed crescentic glomerulonephritis. Immunofluorescence study showed peripheral granular deposits of IgG, IgM, and C3 in the glomeruli. Cresents were predominantly composed of macrophages and glomerular epithelial cells. Amyloid nephropathy, renal vasuculitis, and association of other collagen vascular diseases were negligible for the causative factor. It was suggested that immune complexes were formed in the glomeruli, in which both humoral and cellular immune responses were to be induced, that brought cescents formation in the lesions. Crescentic glomerulonephritis in patients with rheumatoid arthritis is rare and a possible pathogenetic mechanisms involved in the development of renal dysfunction are discussed with the special reference to immune complex-induced inflammation.     

Electrophoretic deposition of carbon nanotubes onto carbon-fiber fabric for production of carbon/epoxy composites with improved mechanical properties

Carbon nanotubes (CNTs) have been deposited onto carbon-fiber fabric using electrophoretic deposition (EPD) prior to the infusion of epoxy resin for the production of carbon/epoxy composites. The carbon-fiber fabric employed for EPD was used in the as-received condition, in which the proprietary epoxy sizing-agent was present. CNTs were functionalized prior to EPD using ozone treatment for oxidation, followed by chemical reaction with polyethyleneimine. The CNT oxidation used a novel recirculating system which enabled ozonolysis to be conducted on large-volume solutions of CNTs in the presence of high-powered sonication, facilitating preparation of stable dispersions suitable for EPD. Significant increases in the shear strength and fracture toughness of the carbon/epoxy composites with the CNT treatment have been measured relative to composites without the CNT treatment. Analysis of fracture surfaces revealed interlaminar regions with high levels of CNTs and evidence of good adhesion between the carbon nanotubes and sized carbon-fiber, which is believed to have contributed to the measured improvement in mechanical properties.     

术前口服依他昔布对妇科手术后病人自控镇痛的影响

目的: 观察妇科开腹手术患者术前给予依他昔布120 mg 对术后自控镇痛(PCA) 吗啡用量的影响及其副作用。方法: 随机选取全麻下接受妇科开腹全子宫(双附件) 切除患者40 例, 术前分别给予安慰剂或依他昔布120 mg 口服。在手术后观察24 h 患者PCA 吗啡用量, 同时记录患者的疼痛评分和满意度评分, 并观察不良反应。结果: 术后PCA 吗啡24h 消耗量在依他昔布组为9.4±7.6 mg, 显著低于安慰剂组15.7±8.9 mg。两组患者24 h 内对PCA 的按压次数依他昔布组也低于安慰剂组(P <0.05)。其中术后3、6、8 和12 h PCA 吗啡消耗量在两组间无显著差异。两组患者术后24 h 疼痛评分和对镇痛治疗的满意度也无差异。两组患者不良反应的发生率亦无差别。结论: 在妇科开腹手术患者术前应用依他昔布120 mg 可使患者术后PCA 吗啡消耗量降低, 且在术后12 h 后下降明显。未发现与依他昔布应用相关的副作用。     

Snowboarding injuries

Snowboarding is a popular winter sport that involves riding a single board down a ski slope or on a half-pipe snow ramp. Compared with injuries resulting from traditional alpine skiing, snowboarding injuries occur more frequently in the upper extremities and ankles and less frequently in the knees. Different types of snowboard equipment, rider stance and snowboarding activity tend to result in different types of injury. Snowboarder's ankle, a fracture of the lateral talus, must be considered in a snowboarder with a "severe ankle sprain" that has not responded to treatment. Risk of injury may be lowered by using protective equipment, such as a helmet and wrist guards.