Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K+ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (PCa/PNa = 5.6 +/- 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, PCa/PNa, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 microM, 240 nM, and 81 microM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed wide-spread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit.     

Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity

PURPOSE: Knowledge of the mechanisms by which retinal ganglion cells are damaged may provide information required to develop novel treatments for diseases that cause retinal ganglion cell death. The authors investigated whether the expression of the 72-kDa heat shock protein in cultured rat retinal ganglion cells increases tolerance to hypoxic and excitotoxic injury. METHODS: Hyperthermia (42 degrees C for 1 hour) and sublethal hypoxia (9% O2 for 6 hours) were used to induce synthesis of the 72-kDa heat shock protein in cultured rat retinal ganglion cells and cultured retinal Müller cells. Induction of the 72-kDa heat shock protein was detected with immunocytochemical and immunoblot techniques. Survival of cultured retinal ganglion cells after exposure to anoxia (< 1% O2 for 6 hours) and glutamate (200 microns for 6 hours) was measured and compared to control cultures stressed previously by hyperthermia or sublethal hypoxia. The effect of quercetin, a blocker of heat shock protein synthesis, was evaluated in parallel experiments. RESULTS: Heat shock protein immunoreactivity was expressed in cultured retinal ganglion cells and Müller cells after hyperthermia and sublethal hypoxia. The mean (+/- standard deviation) retinal ganglion cell survival rates after exposure to anoxia (expressed as a percentage of untreated control cultures) in cells pretreated with sublethal hypoxia (83% +/- 17%) and hyperthermia (82% +/- 19%) were significantly greater than for cells that had no pretreatment (50% +/- 18%, P < 0.001). The mean (+/-standard deviation) retinal ganglion cell survival rate after exposure to glutamate in cells pretreated with sublethal hypoxia (82% +/- 19%) and hyperthermia (86% +/- 17%) were significantly greater than for cells that had no pretreatment (56% +/- 17%, P < 0.001). Inhibition of heat shock protein synthesis with quercetin abolished the protective effects of sublethal hypoxia and hyperthermia on cell survival after anoxia and glutamate exposure. CONCLUSIONS: The neuroprotective effect of hyperthermia and sublethal hypoxia suggests that heat shock proteins confer protection against ischemic and excitotoxic retinal ganglion cell death.     

Kinetic trapping of oxygen in cell respiration

Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase. In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre. It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak. This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre. Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping. Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2. This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration.     

Segregation of germ granules in living Caenorhabditis elegans embryos: cell-type-specific mechanisms for cytoplasmic localisation

Extrinsic allergic alveolitis and pulmonary sarcoidosis are granulomatous diseases of the lung for which clinical presentation and anatomic site of granuloma formation differ. Extrinsic allergic alveolitis is caused by inhaled antigens, whereas the nature and source of the inciting antigen in sarcoidosis is unknown. To test the hypothesis that the route via which antigen is introduced to the lung contributes to the clinicopathological presentation of pulmonary granulomatous disease, rats immunized with intravenous (i.v.) Corynebacterium parvum were challenged after 2 weeks with either intratracheal (i.t.) or i.v. C. parvum. The granulomatous inflammation elicited by i.t. challenge predominantly involved alveolar spaces and histologically simulated extrinsic allergic alveolitis. In contrast, the inflammation induced by i.v. challenge was characterized by granulomatous angiitis and interstitial inflammation simulating sarcoidosis. Elevations of leukocyte counts and TNF levels in bronchoalveolar fluid, which reflect inflammation in the intra-alveolar compartment, were much more pronounced after i.t. than after i.v. challenge. Tumor necrosis factor, interleukin-6, CC chemokine, CXC chemokine, and adhesion molecule mRNA and protein expression occurred in each model. In conclusion, i.t. or i.v. challenge with C. parvum in sensitized rats caused pulmonary granulomatous inflammation that was histologically similar to human extrinsic allergic alveolitis and sarcoidosis, respectively. Although the soluble and cellular mediators of granulomatous inflammation were qualitatively similar in both disease models, the differing anatomic source of the same antigenic challenge was responsible for differing clinicopathological presentations.     

Characterization of type I 5 alpha-reductase activity in DU145 human prostatic adenocarcinoma cells

Each amino acid is represented by a vector of numerical measurements for the attributes of volume, area, hydrophilicity, polarity, hydrogen bonding, shape, and charge. Inter-residue distances are then calculated according to common metrics, and we introduce a new clustering objective function derived from information-theoretic considerations. The arguments of the function are the inter-object distances of the things to be clustered: in this case the amino acids. By means of approximating the solution of an integer programming problem, then, the residues are partitioned into clusters. The clusters obtained are compared with groups obtained in substitution/mutation studies and found to be similar. Thus, probably the strongest and most objective evidence to date is supplied for believing that physico-chemical properties account for the viability of substitutions and that the important similarities/differences are explained by a relatively small and simple set of properties.     

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.     

Protein content and cAMP-dependent phosphorylation of fractionated white perch retina

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.     

Co-morbidities and survival of men with localized prostate cancer treated with surgery or radiation therapy

PURPOSE: We determined the impact of preexisting co-morbidities on survival of men with clinical stages T1b and T2NXM0 prostate cancer treated with surgery or radiation therapy. MATERIALS AND METHODS: A weighted co-morbidity score was determined for 276 consecutive men treated with surgery (138) or radiation therapy (138) at a Veterans Affairs medical center and was correlated with actuarial freedom from death due to co-morbid disease. RESULTS: After a median potential followup of 7.0 years 91 patients (33%) died of co-morbid disease and 20 (7%) died of cancer related causes. There were highly significant correlations between actuarial survival and weighted co-morbidity (p < 0.000001), and the 10-year actuarial survivals in men with no or severe co-morbidities were 66 and 9%, respectively. Associations between patient age and co-morbidity score were highly significant (p < 0.0001). The age adjusted risk of co-morbid death was 5.7 times greater in men with severe compared to no co-morbidities. There were also significant correlations between actuarial survival and weighted co-morbidity among patients treated with surgery (p = 0.02) and radiation therapy (p = 0.0002). Patient age and severity of co-morbidities were significantly greater among men treated with radiation therapy compared to surgery, and age adjusted risk of co-morbid death among men with a co-morbidity score of 1 was 3.8 times greater among men treated with radiation therapy (p = 0.025). CONCLUSIONS: Cancer related deaths are unusual within 5 to 10 years after surgery or radiation therapy in men with stages T1b and 2 prostate cancer. The risk of death during this interval is directly related to the severity of co-morbid conditions, which should be factored in an individual when assessing the advisability of therapeutic intervention. Since patient co-morbidities impact all cause survival, quantitative assessment of co-morbidities using validated instruments offers a method to control partially for the variabilities of health status among men receiving different treatments for localized prostate cancer.     

Altered basal and insulin-stimulated phosphotyrosine phosphatase (PTPase) activity in skeletal muscle from NIDDM patients compared with control subjects

To measure possible changes in basal and insulin-stimulated phosphotyrosine phosphatase (PTPase) activity in skeletal muscle from insulin-resistant individuals, soluble and particulate muscle fractions were prepared from biopsies taken before and after a 3-h hyperinsulinaemic euglycaemic clamp in eight non-insulin-dependent diabetic (NIDDM) patients and nine control subjects. We used a sensitive sandwich-immunofluorescence assay and the human insulin receptor as the substrate. PTPase activity was expressed as percentage of dephosphorylation of phosphotyrosyl-residues in immobilized insulin receptors per 2 h incubation time per 83 micrograms and 19 micrograms muscle fraction protein (soluble and particulate fraction, respectively). In the diabetic soluble muscle fractions, the basal PTPase activity was decreased compared with that of control subjects (11.5 +/- 5.5 vs 27.5 +/- 3.3, p < 0.04, mean +/- SEM). In the particulate muscle fractions from the control subjects, PTPase activity was increased after 3 h hyperinsulinaemia (20.0 +/- 3.2 vs 30.2 +/- 3.6, p < 0.03) and in the corresponding soluble fractions PTPase activity seemed decreased (27.5 +/- 3.3 vs 19.9 +/- 5.9, NS). No effect of insulin on PTPase activity was found in NIDDM patients (25.1 +/- 4.1 vs 27.2 +/- 5.2, 11.5 +/- 5.5 vs 15.1 +/- 4.5 [particulate and soluble fractions], NS). In conclusion, we found that the basal PTPase activity in soluble muscle fractions was decreased in NIDDM patients; furthermore, insulin stimulation was unable to increase PTPase activities in the particulate fractions, as opposed to the effect of insulin in control subjects.