Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation

High density lipoproteins (HDL, d 1.063-1.21 g/ml) are reported to stimulate, to have no effect on, or to inhibit agonist-induced platelet aggregation. We have hypothesized that these conflicting reports might be explained by opposing effects of individual HDL subclasses on platelet aggregability. Physiologic concentrations of HDL3 had little effect on ADP-induced aggregation of washed platelet suspensions, although higher levels were stimulatory. Normal concentrations of HDL2 (0.2-0.4 mg of protein/ml) inhibited aggregation; further fractionation by heparin-Sepharose chromatography identified the particles rich in apolipoprotein E, termed HDL-E, as the major anti-aggregatory subclass. Washed platelets bound radioiodinated HDL-E to a uniform class of saturable sites; they numbered 4,200 per platelet and the KD was 7.9 x 10(-7) M. Binding of HDL-E by platelets, and its anti-aggregatory action, showed a similar rapidity and both occurred within the physiologic concentration range. Moreover, the two processes were independent of the presence of divalent ions and were impaired by chemical modification of the apolipoprotein constituents of HDL-E. We conclude that occupation of cell-surface receptors by HDL-E particles impairs platelet responsiveness to exogenous agonists and that platelet aggregability in the presence of whole HDL may reflect the relative concentrations of the individual subclasses in the particular sample.     

The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.     

Immunogold localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in the nitrogen-fixing cyanobacterium,Plectonema boryanum

Summary Antiserum against the Calvin cycle enzyme, ribulose-1,5-bisphosphate carobxylase/oxygenase (RuBisCO), was used in conjunction with colloidal gold to localize RuBisCO in nitrogen-fixing (fix+) and nonfixing (fix–)Plectonema boryanum cells. RuBisCO antiserum consistently labeled the cytoplasm and polyhedral bodies (carboxysomes) in both fix+ and fix– cells. Through morphometry, it was determined that significantly less gold label (indicative of RuBisCO) was present in fix+ cells. This decreased RuBisCO content correlated with a decrease in net photosynthetic oxygen evolution also observed in fix+P. boryanum.Abbreviations RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase - fix+ nitrogen-fixing - fix– nonfixing     

Experience of MOET with Welsh Black cattle in a group breeding scheme

The embryo production records of 27 Welsh Black cows in a multiple ovulation and embryo transfer (MOET) program were examined. Signlficant monthly variations in the number of viable embryos recovered (P=0.07) and in embryo viability (P=0.002) were detected, although ovarian responses did not vary. Embryo recovery was not affected by the type of catheter used or by the side of the uterus flushed.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996