Plasminogen activator regulation in osteoblasts: parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA.

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone.     

Development of spinal motor networks in the chick embryo.

We have examined the cellular and synaptic mechanisms underlying the genesis of alternating motor activity in the developing spinal cord of the chick embryo. Experiments were performed on the isolated lumbosacral cord maintained in vitro. Intracellular and whole cell patch clamp recordings obtained from sartorius (primarily a hip flexor) and femorotibialis (a knee extensor) motoneurons showed that both classes of cell are depolarized simultaneously during each cycle of motor activity. Sartorius motoneurons generally fire two bursts/cycle, whereas femorotibialis motoneurons discharge throughout their depolarization, with peak activity between the sartorius bursts. Voltage clamp recordings revealed that inhibitory and excitatory synaptic currents are responsible for the depolarization of sartorius motoneurons, whereas femorotibialis motoneurons are activated principally by excitatory currents. Early in development, the dominant synaptic currents in rhythmically active sartorius motoneurons appear to be inhibitory so that firing is restricted to a single, brief burst at the beginning of each cycle. In E7-E13 embryos, lumbosacral motor activity could be evoked following stimulation in the brainstem, even when the brachial and cervical cord was bathed in a reduced calcium solution to block chemical synaptic transmission. These findings suggest that functional descending connections from the brainstem to the lumbar cord are present by E7, although activation of ascending axons or electrical synapses cannot be eliminated. Ablation, optical, and immunocytochemical experiments were performed to characterize the interneuronal network responsible for the synaptic activation of motoneurons. Ablation experiments were used to show that the essential interneuronal elements required for the rhythmic alternation are in the ventral part of the cord. This observation was supported by real-time Fura-2 imaging of the neuronal calcium transients accompanying motor activity, which revealed that a high proportion of rhythmically active cells are located in the ventrolateral part of the cord and that activity could begin in this region. The fluorescence transients in the majority of neurons, including motoneurons, occurred in phase with ventral root or muscle nerve activity, implying synchronized neuronal action in the rhythm generating network. Immunocytochemical experiments were performed in E14-E16 embryos to localize putative inhibitory interneurons that might be involved in the genesis or patterning of motor activity. The results revealed a pattern similar to that seen in other vertebrates with the dorsal horn containing neurons with gamma-aminobutyric acid (GABA)-like immunoreactivity and the ventral and intermediate regions containing neurons with glycine-like immunoreactivity.     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water.

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

A Canadian hospital's experience with the automatic implantable cardioverter/defibrillator.

The automatic implantable cardioverter/defibrillator is a device that can be implanted in patients for treatment of recurrent ventricular tachycardia and ventricular fibrillation. It was recently approved for clinical use in Canada. The authors describe their experience with 12 patients (mean age 51.3 years) who underwent implantation of a defibrillator. All 12 patients had a history of documented ventricular fibrillation, which was idiopathic in 3 and due to ischemic heart disease in 9. Electrophysiologic testing revealed inducible ventricular tachycardia or ventricular fibrillation in 8 of the 10 patients tested. An important criterion for selection for implantation was failure of pharmacologic therapy to suppress ventricular arrhythmias induced during electrophysiologic testing. Of the 12 patients, 1 died within 24 hours after implantation. During a mean follow-up period of 15.5 months there were no further deaths. All the surviving patients expressed satisfaction with the device; five of the seven under the age of 60 years have returned to work, and one has returned to school. This initial favourable experience with the automatic implantable cardioverter/defibrillator suggests that future increases in the availability of the device and improvements in its function will lead to much more widespread use, as the population of patients at risk of sudden cardiac death is large.