Phosphate transport in osteoblasts from normal and X-linked hypophosphatemic mice

Human hypophosphatemic vitamin D-resistant rickets (X-linked hypophosphatemia-XLH) is characterized by hypophosphatemia, a decreased tubular reabsorption of phosphate (P_i) and defective skeleton mineralization. Utilizing a mouse model (Hyp) of XLH, which demonstrates biological abnormalities and skeletal defects of XLH, we analyzed sodium-dependent phosphate transport in isolated osteoblasts derived from the calvaria of normophosphatemic and hypophosphatemic mice. Initial rates of phosphate uptake by normal and Hyp osteoblasts showed similar slopes. Osteoblasts from both normal and Hyp mice exhibited saturable, sodium-dependent phosphate transport with apparent V_max and K_m values not significantly different (normal mice, V_max=24.30±3.45 nmol/mg prot. 10 min, K_m=349.49±95.20 mol/liter; Hyp mice, V_max=23.03±3.41 nmol/mg prot. 10 min, K_m=453.64±106.93 mol/liter, n=24). No differences were found in the ability of normal and Hyp osteoblasts to respond to P_i transport after 5 hours of P_i deprivation. Both cell types exhibited a similar increase in cAMP in response to PTH. The accumulated results demonstrate that P_i uptake and transport in normal and Hyp mouse osteoblasts is a sodium-dependent saturable process. As osteoblast P_i uptake and transport is apparently normal in the Hyp mouse model of XLH, the osteoblastic failure described for the Hyp mouse should be attributed to other mechanism(s).     

Defective RNA molecules associated with citrus tristeza virus

Preparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5' (1151 nt) and the 3' (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5' or 3' terminus. This is the first defective RNA to be reported for a member of the closterovirus group.     

Third-trimester amniotic fluid metal levels associated with preeclampsia

Studies of maternal amniotic fluid and serum at delivery have revealed the presence of toxic metals or deficiencies of essential metals associated with high-risk pregnancy. Essential and toxic metal levels were studied in 29 preeclamptic and 101 normal pregnancies. The authors grouped the samples according to the following gestation ages: 33-36 wk (48 normal and 10 preeclamptic) and 37-40 wk (53 normal and 19 preeclamptic). The metals studied were calcium, magnesium, zinc, selenium, copper, cadmium, and lead. Comparisons of the 33-36-wk gestation group showed significant differences between normal and preeclamptic amniotic fluid in levels of lead, calcium, magnesium, zinc, and selenium. There were also significant differences in levels of magnesium, copper, zinc, cadmium, and lead during the gestation period of 37-40 wk. The changes observed in calcium and lead levels were consistent with the results of animal studies in which investigators have found depressed calcium transport associated with subacute or chronic lead poisoning, resulting in a physical syndrome similar to preeclampsia.     

Generation of oligodendroglial progenitors in acute inflammatory demyelinating lesions of the rat brain stem is associated with demyelination rather than inflammation

Remyelination is an extremely efficient process in the adult rodent central nervous system yet the source of new oligodendroglia that appear following primary demyelination is still subject to much debate. Using a reliable marker for oligodendroglial progenitor cells in vivo, the NG2 chondroitin sulphate proteoglycan, we have evaluated the response of endogenous NG2⁺ cells in the adult rat brain stem and cerebellum to inflammatory demyelinating lesions in an experimentally induced animal model of multiple sclerosis (MS), antibody augmented experimental allergic encephalomyelitis (ADEAE). We have manipulated T-cell mediated EAE in Lewis rats by injecting in addition, either anti-myelin/oligodendroglial glycoprotein (MOG) antibodies to induce inflammatory demyelination, or non-specific mouse immunoglobulins to induce an inflammatory response without demyelination. We have examined the relationship of NG2⁺ progenitor cells to microglia (OX-42⁺), astrocytes (GFAP⁺) and mature oligodendroglia (CNP⁺), in the normal and demyelinated CNS. In the normal CNS NG2-expressing cells are closely intermingled with other glia but represent a distinct cell population. A prominent inflammatory response, identified by the presence of large perivascular and periventricular accumulations of reactive OX42⁺ macrophages/microglia, occurred in animals with ADEAE at 7–9 days post injection (DPI), coinciding with severe clinical symptoms. In animals injected with anti-MOG antibodies inflammation was followed by the appearance of large areas of demyelination at 11–14 DPI, at which point the animals had recovered clinically. The response of NG2⁺ cells was different depending on whether the inflammation was accompanied by demyelination. In the presence of inflammation, NG2⁺ cells responded by an increase in immunoreactivity and an alteration in their morphology, exhibiting enlarged cell bodies and an increased number of intensely stained processes. In areas of demyelination NG2⁺ cells had fewer intensely stained processes reminiscent of progenitor cells seen during development. Quantitative analysis revealed a 3-fold increase in the number of NG2⁺ cells in demyelinated lesions at 11 DPI, whereas no change was observed in areas of inflammation in the absence of demyelination. Mitotic figures were only seen in NG2⁺ cells in areas of demyelination. NG2⁺ cell numbers appeared to return to control levels following remyelination. These results suggest that endogenous oligodendroglial progenitors divide and/or migrate, in response to signals triggered by demyelinating rather than inflammatory events, to generate a large progenitor population sufficient to promote the rapid and successful remyelination observed in this model.     

Mild traumatic brain injury from motor vehicle accidents: factors associated with return to work

OBJECTIVES: To describe return to work (RTW) for motor vehicle accident (MVA) survivors with mild traumatic brain injury (MTBI) and to examine relationships between RTW and injury severity, cognitive impairment, social interaction, discharge disposition, and sociodemographics. DESIGN: Inception cohort assessed within 1 month of injury and at follow-up 6 to 9 months (mean = 7.4) after injury, for comparisons on outcome of RTW. SETTING: Tertiary care center in Toronto (time 1); at home for follow-up. PARTICIPANTS: Fifty patients with MTBI resulting from MVA who were consecutively admitted during a 20-month period ending April 1994. Thirteen of 63 eligible patients refused consent or were lost to follow-up. Mean age was 31; 62% were men. Eligibility criteria: (1) patients had been working; (2) they had no history of head injury, neurologic disease, or psychiatric illness requiring hospitalization; and (3) they had no catastrophic impairment from accident. MAIN OUTCOME MEASURE: Return to work (at premorbid or modified level). RESULTS: Of the 42% who returned to work, 12% resumed their premorbid level of employment and 30% returned to modified work. There were significant differences (p<.05) between the groups in level of social interaction, premorbid occupation, and discharge disposition. On one test of cognitive functioning the difference was at p = .06. CONCLUSION: Social interaction, jobs with greater decision-making latitude, and discharge home were positively related to RTW for this population. Cognitive impairment within the first month was not a reliable indicator of RTW potential.     

Evaluation of [methyl-3H]L655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABA(A) receptors containing alpha5 subunit

Two selective radioligands of gamma aminobutyric acid (GABA)A receptors containing the alpha5 subunit, [3H]L655,708 and [3H]RY80, were evaluated in rats as potential in vivo tracers for positron emission tomography (PET). Brain uptake index (BUI), a measure of first pass extraction, was moderate for [3H]L655,708 (BUI of 59%) and good for [3H]RY80 (BUI of 96%). This finding was consistent with their in vitro binding to plasma proteins of approximately 76% and 50%, respectively. Following intravenous injection of either radioligand, radioactivity in plasma was measured and uptake characteristics were assessed in brain within a time period relevant to PET scanning (up to 90 min). Discrete brain regions, such as frontal cortex, striatum, hypothalamus, thalamus, hippocampus, colliculi, medulla, and cerebellum, were sampled and the temporal distribution of radioactivity analysed. Despite the reasonable delivery to the brain, neither of the radioligands had sufficient retention in the tissues rich in alpha5-containing GABA(A) receptors to achieve a good selective signal. For both radioligands, a maximal tissue:cerebellum ratio of 1.5 was seen in hippocampus at 10 min after injection. Thus, neither of the compounds studied shows potential for further development as an in vivo PET ligand.     

Differential responses of oligodendrocytes to tumor necrosis factor and other pro-apoptotic agents: role of ceramide in apoptosis

Staurosporine induced apoptosis in a human oligodendroglioma cell line (HOG), neonatal rat oligodendrocyte (O2A(+)) precursors, and mature rat oligodendrocytes. In all three cell culture systems, the activation of caspase-3-like activity (CPP32) coincided with the increased formation of ceramide from sphingomyelin and the onset of DNA fragmentation. Further, the addition of exogenous C(2)-ceramide induced CPP32 activation and DNA fragmentation in all three culture systems. Raising endogenous ceramide levels by the addition of the ceramidase inhibitor, oleoylethanolamine, enhanced apoptosis in both a time- and concentration-dependent manner. Inhibitors of phosphatidylinositol 3-kinase (wortmannin and LY294002) also induced caspase-3 (CPP32) activation, increased ceramide formation, induced DNA fragmentation, and reduced cell viability. In contrast, cytokines such as tumor necrosis factor-alpha (TNF-alpha) had a differential effect on the three cell cultures. Thus, TNF-alpha (160 ng/ml) induced 70% apoptosis in 24 hr in freshly isolated rat brain O2A(+) precursor cells, 60% apoptosis in 24 hr in a human oligodendroglioma (HOG) cell line, but no apoptosis in mature neonatal rat oligodendrocytes. Interferon-gamma augmented the activation of CPP32 by TNF-alpha in HOG cells and O2A(+) oligodendrocyte precursor cells but had no effect on mature oligodendrocytes. Thus, the death pathway appears to be similar in the three cell lines but the lack of coupling between TNF-alpha receptors and the apoptotic pathway leads to a lack of response to cytokines in mature oligodendrocytes.     

Synthetic retinoid CD437 induces S-phase arrest and apoptosis in human prostate cancer cells LNCaP and PC-3

BACKGROUND: Exposure of prostate carcinoma cell lines to retinoids, which function through the classical retinoic acid nuclear receptor, (RARs) or retinoid X receptors (RXRs), results in minimal cytostatic inhibition of cell proliferation. METHODS: Growth inhibition and various regulatory responses were investigated in two human prostate carcinoma cell lines (LNCaP and PC-3) treated with or without a synthetic retinoid, CD 437. RESULTS: Incubation of prostate carcinoma cell lines with a novel retinoid CD437 resulted in the marked inhibition of proliferation. LNCaP and PC-3 possessed IC50 values for CD437 of 375 nM and 550 nM, respectively. Incubation with 1 microM CD437 for 24 hr resulted in 100% and 60% inhibition of growth in LNCaP and PC-3 cells, respectively. Simultaneously, cell flow cytometric analyses revealed a dramatic increase of the cell population in S phase, in both LNCaP (from 38.6% up to 86.7%) and PC-3 (27.9% to 55.7%), and a decreased proportion of cells in G2 phase, in LNCaP (from 23.7% down to 1.2%) and PC-3 (14.9% to 2.2%), indicating a significant S-phase arrest. The cell growth inhibition and S-phase arrest in these cells were followed by apoptosis, as revealed by the acquisition of the characteristic cell morphology including the appearance of apoptotic bodies, and further confirmed by cellular DNA fragmentation. CD437-induced-S phase arrest was associated with upregulated mRNA levels of p21waf1/cip1/sdi1 in both LNCaP (p53+/+) and PC-3 (53-/-) cells. CONCLUSIONS: CD437 represents a unique retinoid that induces S-phase arrest and apoptosis in both androgen-dependent (LNCaP) and -independent (PC-3) human prostate cancer cells, suggesting a potential role of CD437 in the treatment of human prostate cancer.     

The assessment of shoulder instability. The development and validation of a questionnaire

We have developed a 12-item questionnaire for completion by patients presenting with shoulder instability. A prospective study of 92 patients was undertaken involving two assessments, approximately six months apart, performed in an outpatient department. Each patient completed the new questionnaire and the SF36 form. An orthopaedic surgeon completed the Constant shoulder score and the Rowe assessment. The new questionnaire and the Rowe clinical score each achieved a large standardised effect size (> or = 0.8) and compared favourably with relevant items on the SF36. By contrast, the Constant score barely registered any effect, confirming that it may be relatively insensitive to changes in clinical status for this particular condition. The questionnaire provides a measurement of outcome for shoulder instability which is short, practical, reliable, valid and sensitive to changes of clinical importance.