OCRL controls trafficking through early endosomes via PtdIns4,5P₂-dependent regulation of endosomal actin

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P(2)) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P(2) in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P(2) and F-actin at the EEs is essential for exporting cargoes that transit this compartment.     

Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin.

We have studied in mice the effect of treatment with exogenous arginine and/or LPS by monitoring serum nitrite/nitrate levels and by investigating the response of cerebellar and liver nitric oxide synthase (NOS). We measured NOS activity in cerebellar extracts while changes in iNOS mRNA were followed in the liver since direct assay of NOS activity proved unreliable with this tissue. In fact, liver and cerebellum extracts were both very active in converting arginine into a citrulline-like metabolite, but only cerebellum conversion was dependent on addition of NADPH and inhibitable by N(G)-methyl-l-arginine. Treatment with LPS, on its own, increased serum nitrite/nitrate levels at 5 and 20 h after injection, while treatment with LPS and arginine produced nitrite/nitrate levels in the serum even greater at 5 h, but significantly lower at 20 h. Liver iNOS mRNA levels were markedly increased by LPS, and this effect was significantly decreased when mice were also given exogenous arginine. A stimulatory effect of LPS was also found on NOS activity in the cerebellum, where a very small stimulation may have also been caused by arginine feeding. These findings indicate that LPS stimulates NOS expression/activity both in the cerebellum and in the liver and suggest a complex pattern of modulation of iNOS by arginine, with NO being first produced in excess and then downregulating iNOS expression.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.     

Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.     

The effect of N-methylprotoporphyrin and succinyl-acetone on the regulation of heme biosynthesis in chicken hepatocytes in culture

The essential features of hepatic protoporphyria, namely inhibition of ferrochelatase, accumulation of protoporphyrin and stimulation of 5-aminolevulinic acid synthase (ALA-S) were all obtained by treating chicken hepatocytes in culture with small doses of N-methylprotoporphyrin. Both N-methylprotoporphyrin and succinyl-acetone, another inhibitor of heme biosynthesis, stimulated ALA-S when given on their own and also enhanced the stimulation of ALA-S caused by phenobarbital.     

The expression analysis of mouse interleukin-6 splice variants argued against their biological relevance

Alternative splicing generates several interleukin-6 (IL-6) isoforms; for them an antagonistic activity to the wild-type IL-6 has been proposed. In this study we quantified the relative abundance of IL-6 mRNA isoforms in a panel of mouse tissues and in C2C12 cells during myoblast differentiation or after treatment with the Ca(2+) ionophore A23187, the AMP-mimetic AICAR and TNF-α. The two mouse IL-6 isoforms identified, IL-6δ5 (deletion of the first 58 bp of exon 5) and IL-6δ3 (lacking exon 3), were not conserved in rat and human, did not exhibit tissue specific regulation, were expressed at low levels and their abundance closely correlated to that of full-length IL-6. Species-specific features of the IL-6 sequence, such as the presence of competitive 3' acceptor site in exon 5 and insertion of retrotransposable elements in intron 3, could explain the production of IL-6δ5 and IL-6δ3. Our results argued against biological significance for mouse IL-6 isoforms.     

Paracentric inversion of chromosome 15(q15q24): description of three families

Three unrelated families with paracentric inversion of chromosome 15(q15q24) are reported. An additional pericentric inversion of chromosome 9 with breakpoints in p11.2q13 was also observed in one of the three families. Reproductive problems, such as stillbirths, spontaneous abortions and two live-born children with multiple abnormalities, were present.