Amphiphysin 1 is important for actin polymerization during phagocytosis

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (-/-) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate-induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.     

Intracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Two soluble glycoproteins containing hydroxyproline were extractedfrom cultured tobacco cells (cell line XD-6S) and purified byion-exchange and gel-filtration chromatography. On DEAR-cellulosecolumn chromatography in the final step of the purification,one was eluted at 90 mM NaCl and the other at 120 mM as singlepeak. Both purified glycoproteins were also sedimented as singlepeak with an ultracentrifugation. The S_20,w values were 6.1for the former and 7.0 for the latter. These glycoproteins were composed of 94% polysaccharide and6% protein in the former, and 87% polysaccharide and 13% proteinin the latter. The sugar moiety consisted of galactose, arabinose,rhamnose, and uronic acid in both. Hydroxyproline accountedfor 12% in the former and 20% in the latter amino acid composition.A high content of alanine in both (14 and 15%) was one of thedistinctive characteristics of these soluble glycoproteins. These intracellular soluble hydroxyproline-containing glycoproteinswere not labelled within 30 min of incubation with ₃H-proline,although the radioactivity was rapidly incorporated (within15 min) into the intracellular macromolecules. (Received February 21, 1978; )     

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.     

Differential metabolism of 4-n- and 4-tert-octylphenols in perfused rat liver

Octylphenols, widely used in a variety of detergents and plastics, are known to exhibit estrogenicity in vivo. The details of their metabolism are needed to better understand the endocrine disruptions. We have previously shown that alkylphenols, having short alkyl chains, are glucuronidated and readily excreted into the bile from the liver, while 4-n-nonylphenol, having longer alkyl chains, remains as the alkylphenol's glucuronide in the tissue. This study elucidated the dependence of the metabolism on the shape of the alkyl chains by comparing 4-n-octylphenol and 4-tert-octylphenols in a perfused rat liver. Both octylphenols were highly glucuronidated by the liver microsomal fractions. The Vmax value of 4-tert-octylphenol glucuronidation was twice as high as that of 4-n-octylphenol in the liver microsomes. On the other hand, the Km values, being measures of enzymatic activity against these chemicals, were similar. 4-n-Octylphenol and 4-tert-octylphenol were both glucuronidated by a UDP-glucuronosyltransferase isoform, UGT2B1, expressed in the liver. In the liver perfusion, almost all of the 4-n-octylphenol perfused was metabolized directly to the glucuronide, whereas a portion of 4-tert-octylphenol was hydroxylated and then glucuronidated. The glucuronide of 4-n-octylphenol accumulated in the liver tissue in the same manner as 4-n-nonylphenol, but 4-tert-octylphenol and the hydroxylated metabolites were excreted readily into the bile. Only a small amount of 4-n-octylphenol-glucuronide and glucuronides of 4-tert-octylphenol and its hydroxylated metabolites could be excreted into the bile of Eisai hyperbilirubinemic rats (EHBR). These animals are deficient in xenobiotic conjugate transporter, multidrug resistance-associated protein (MRP-2), indicating that the glucuronides of both octylphenols are transported by MRP-2. These results indicate that the differences in metabolism of these octylphenols are due to the shape of their alkyl chains, suggesting that the estrogenic activities of not only the parent chemicals but also these metabolites must be taken into consideration.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

Triterpenoid saponins from Fatsia japonica

Five triterpenoid saponins isolated from the flowers, the mature fruits and the leaves of Fatsia japonica were identified as 3-O-[β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl]-hederagenin (1), 3-O-[β-d-glucopyranosyl-(1→4)-α-l-arabinopyranosyl]-oleanolic acid (2), 3-O-[α-l-arabinopyranosyl]-hederagenin (3), 3-O-[β-d-glucopyranosyl]-hederagenin (4) and 3-O-[β-d-glucopyranosyl(1→4)-α-l-arabinopyranosyl]-hederagenin (5). The saponins 1 and 2 are new, naturally occurring, triterpenoid saponins. The distribution of the five saponins in three parts of the plant was investigated. Saponins 2, 3 and 5 were present in the flowers, saponins 1, 3, 4 and 5 were in the mature fruits and saponins 2, 3, 4 and 5 were in the leaves.     

Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data

Lathyrus (Leguminosae; Papilionoideae) is the largest genus in tribe Fabeae and exhibits an intriguing extratropical distribution. We studied the systematics and biogeography of Lathyrus using sequence data, from accessions representing 53 species, for the internal transcribed spacer plus 5.8S-coding region of nuclear ribosomal DNA as well as the trnL-F and trnS-G regions of chloroplast DNA. Our results generally supported recent morphology-based classifications, resolving clades corresponding to sections Lathyrus and Lathyrostylis, but question the monophyly of the large, widespread section Orobus sensu Asmussen and Liston. Sections Orobus, Aphaca, and Pratensis form a predominantly northern Eurasian-New World clade. Within this clade, the North American and eastern Eurasian species, including both Holarctic species (L. palustris and L. japonicus), form a transberingian clade of relatively recent origin and diversification. The South American Notolathyrus group is distant from this transberingian lineage and should be reinstated as a distinct section within the northern Eurasian-New World clade. The Notolathyrus lineage reached the New World most probably through long-distance dispersal from Eurasia. The remaining sections in the genus are centered on the Mediterranean region.     

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.     

Ruptured renal arteriovenous malformation successfully treated by catheter embolization: a case report

Background

Fabry disease is an X-linked inherited metabolic condition where the deficit of the α-galactosidase A enzyme, encoded by the GLA gene, leads to glycosphingolipid storage, mainly globotriaosylceramide. To date, more than 600 mutations have been identified in human GLA gene that are responsible for FD, including missense and nonsense mutations, small and large deletions. Such mutations are usually inherited, and cases of de novo onset occur rarely.

Case presentation

In this article we report an interesting case of a 44-year-old male patient suffering from a severe form of Fabry disease, with negative family history. The patient showed signs such as cornea verticillata, angiokeratomas, cardiac and neurological manifestations, an end-stage renal disease and he had low α-galactosidase A activity. We detected, in this subject, the mutation c.493 G?>?C in the third exon of the GLA gene which causes the amino acid substitution D165H in the protein. This mutation affects the amino acid - belonging to the group of buried residues - involved, probably, in the preservation of the protein folding. Moreover, studies of multiple sequence alignment indicate that this amino acid is highly conserved, thus strengthening the hypothesis that it is a key amino acid to the enzyme functionality. The study of the relatives of the patient showed that, surprisingly, none of the members of his family of origin had this genetic alteration, suggesting a de novo mutation. Only his 11-year-old daughter - showing acroparaesthesias and heat intolerance with reduced enzymatic activity - had the same mutation.

Conclusions

We suggest that a non-inherited mutation of the α-galactosidase A gene is responsible for Fabry disease in the patient who had reduced enzyme activity and classical clinical manifestations of the disease. In a family, it is rare to find only one Fabry disease affected subject with a de novo mutation. These findings emphasize the importance of early diagnosis, genetic counselling, studying the genealogical tree of the patients and starting enzyme replacement therapy to prevent irreversible vital organ damage that occurs during the course of the disease.