Calmodulin antagonists inhibit Ca2+ uptake of mitochondria of guinea pig peritoneal macrophages

The Ca²⁺ uptake of the mitochondria of guinea pig peritoneal macrophages was not stimulated by the addition of calmodulin. However, calmodulin antagonists, both phenotiazines and N-naphthalenesulfonamides, in low concentrations inhibited the Ca²⁺ uptake of the mitochondoria, as compared to the inhibition of the calmodulin-dependent stimulation of brain phosphodiesterase. These calmodulin antagonists appear to have severe side effects on active processes of the mitochondria and which are unrelated to the specific effect on calmodulin.     

ATP-dependent Ca2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca²⁺ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{4 × 10}{}^6$ macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10⁶ M^?1. In the absence of sodium azide, the value of Ca²⁺ uptake of saponin-treated macrophages was 95 nmoles/4 × 10⁶ macrophages, and its affinity constant for Ca²⁺ was 3 × 10⁵ M^?1. Saponin-treated macrophages may be suitable for the studies of Ca²⁺ transport systems in the intracellular membranes.     

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl^?, Br^?>I^?>SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$. After 30-min incubation, the extent of aggregation of platelets in Cl^? or Br^? medium was little changed, whereas, that in SCN^? or $\text{ClO}{}_4{}^{\mathrm{?}}$ medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.     

Stimulation of Ca efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10⁻¹⁰ M to 10⁻⁷ M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

An A alpha Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.

We have identified a unique N-glycosylated Asn substitution for a Ser at position 434 of the A alpha chain of an abnormal fibrinogen designated fibrinogen Caracas II. This dysfibrinogen was characterized by impaired fibrin monomer aggregation. Since there were 4 Thr residues immediately following the mutation, a new Asn-X-Thr/Ser-type consensus sequence, Asn-Thr-Thr arose for N-glycosylation of the Asn. The extra oligosaccharide was found to consist mainly of a disialylated biantennary structure comprising 81.9%, while a neutral and a monosialylated biantennary oligosaccharide represented only 3.6% and 14.5%, respectively. The mutation resides in the carboxyl-terminal region of the A alpha chain, which could fold back to form an extra small globular region located near the central region of the molecule (Erickson, H.P., and Fowler, W.E. (1983) Ann. N. Y. Acad. Sci. 408, 146-163; Weisel, H.P., Stauffacher, C.V., Bullitt, E., and Cohen, C. (1985) Science 230, 3124-3133). Therefore, the participation of this region, referred to as an additional central domain or an alpha domain, in fibrin gel formation is strongly implicated.     

A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae

The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.     

Oxidized low density lipoprotein inhibits bradykinin-induced phosphoinositide hydrolysis in cultured bovine aortic endothelial cells.

Vascular endothelial cells, in response to various neurohumoral and physical stimuli, produce an endothelium-derived relaxing factor, a substance which regulates vascular tone. We have demonstrated that oxidized low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. We studied the effect of oxidized LDL on inositol phosphates formation stimulated with bradykinin (BK) in cultured bovine aortic endothelial cells. BK elicited a rapid generation of inositol phosphates from inositol phospholipids. Accumulation of inositol 1,4,5-trisphosphate (IP3) stimulated with BK (0.1 microM) was markedly inhibited by oxidized LDL. However, native LDL had little effect on BK-induced accumulation of IP3. From these results, oxidized LDL inhibits receptor-mediated phosphoinositides hydrolysis and modulates the endothelial function.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.