Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Degradation of o-methoxybenzoate by a two-member consortium made up of a gram-positive Arthrobacter strain and a gram-negative Pantotea strain

Aromatic carboxylic acids substituted with methoxylated groupsare among the most abundant products in alpechin, the wastes resulting from pressing olives to obtain olive oil. Degradation of o-methoxybenzoate by an stable consortium made of a grampositive bacterium, Arthrobacter oxydans, and gram negative one,Pantotea agglomerans, was shown to mineralize this compound efficiently. he concerted action of both microorganisms was needed for the two first steps n the process, namely, the conversion of o-methoxybenzoate into salycilate,and the hydroxylation of the latter to gentisate. Gentisate was further degraded by the Arthrobacter strain.     

Thromboxane-B(2) levels in serum of rabbits receiving a single intravenous dose of aqueous extract of garlic and onion

We have shown previously that fresh garlic extract is effective in reducing thromboxane formation by platelets both in vivo and in vitro animal models of thrombosis. In the present study, the effect of different concentrations of a single dose of aqueous extracts of garlic and onion were evaluated on serum thromboxane-B(2)synthesis in rabbits. Different concentrations of garlic and onion were administered as single doses in the ear vein of rabbits. Rabbits were bled before and at different intervals after the infusion of garlic or onion extracts. Venous blood was collected and allowed to clot at 37 degrees C for 1 h. Thromboxane-B(2)level was measured in the serum by radioimmunoassay. It was observed that garlic inhibits the thrombin-induced platelet synthesis of TXB(2)in a dose-and time-dependent manner. Maximum inhibition of TXB(2)occurred between 0.5 h and 6 h at 25 and 100 mg kg(-1)garlic. At 24 h post-garlic infusion TXB(2)inhibition was reduced to 15% of the control and TXB(2)levels were comparable to that of the control values at 72 h pots-garlic infusion. Infusion of 100 mg kg(-1)onion extract did not elicit any inhibitory effect on TXB(2)synthesis in the serum of rabbit during the treatment period. The rapid recovery of platelet cyclooxygenase activity after infusion of a single dose of garlic suggests that garlic should be taken more frequently in order to achieve beneficial effects in the prevention of thrombosis.     

Detoxication of the herbicide diuron byPseudomonas sp.

A strain of bacteria able to detoxicate the herbicide diuron in pure culture was isolated from sites contaminated with different urea herbicides. Diuron was used as a sole source of carbon and energy by this isolate which is a Gram-negative, aerobic, rod-shaped bacterium with a single polar flagellum, and grows at 40 degrees C. The strain has been identified as Pseudomonas sp.     

Molecular analysis of 16 Turkish families with DHPR deficiency using denaturing gradient gel electrophoresis (DGGE)

Dihydropteridine reductase (DHPR) catalyses the conversion of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4), which serves as the obligatory cofactor for the aromatic amino acid hydroxylases. DHPR deficiency, caused by mutations in the QDPR gene, results in hyperphenylalaninemia and deficiency of various neurotransmitters in the central nervous system, with severe neurological symptoms as a consequence. We have studied, at the clinical and molecular levels, 17 patients belonging to 16 Turkish families with DHPR deficiency. The patients were detected at neonatal screening for hyperphenylalaninemia or upon the development of neurological symptoms. To identify the disease causing molecular defects, we developed a sensitive screening method that rapidly scans the entire open reading frame and all splice sites of the QDPR gene. This method combines PCR amplification and "GC-clamping" of each of the seven exonic regions of QDPR, resolution of mutations by denaturing gradient gel electrophoresis (DGGE), and identification of mutations by direct sequence analysis. A total of ten different mutations were identified, of which three are known (G23D, Y150C, R221X) and the remaining are novel (G17R, G18D, W35fs, Q66R, W90X, S97fs and G149R). Six of these mutations are missense variants, two are nonsense mutations, and two are frameshift mutations. All patients had homoallelic genotypes, which allowed the establishment of genotype-phenotype associations. Our findings suggest that DGGE is a fast and efficient method for detection of mutations in the QDPR gene, which may be useful for confirmatory DNA-based diagnosis, genetic counselling and prenatal diagnosis in DHPR deficiency.     

A microsomal GTPase is required for glycopeptide export from the mammalian endoplasmic reticulum

Bidirectional transport of proteins via the Sec61p translocon across the endoplasmic reticulum (ER) membrane is a recognized component of the ER quality control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported from the ER lumen back to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eukaryotic phenomenon. Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established system to study glycopeptide export and conventional liquid chromatography, we isolated a single polypeptide species of 23 kDa from rat liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by mass spectrometric sequence analysis as guanylate kinase (GK), a housekeeping enzyme critical in the regulation of cellular GTP levels. We confirmed the ability of GK to substitute for complete cytosol by reconstitution of glycopeptide export from rat liver microsomes using highly purified recombinant GK from Saccharomyces cerevisiae. Most significantly, we found that the GK (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a requirement for GTP hydrolysis. Membrane integrity was fully maintained under assay conditions, as no ER luminal proteins were released. Competence for glycopeptide export was abolished by very mild protease treatment of microsomes, indicating the presence of an essential protein on the cytosolic face of the ER membrane. These data demonstrate that export of glycopeptide export is controlled by a microsomal GTPase and is independent of cytosolic protein factors.     

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.     

Glossina austeni (Diptera: Glossinidae) eradicated on the island of Unguja, Zanzibar, using the sterile insect technique

An area-wide integrated tsetse eradication project was initiated in Zanzibar in 1994 by the International Atomic Energy Agency and the governments of Tanzania and Zanzibar, to eradicate Glossina austeni Newstead from Unguja Island (Zanzibar) using the sterile insect technique. Suppression of the tsetse population on Unguja was initiated in 1988 by applying residual pyrethroids as a pour-on formulation to livestock and by the deployment of insecticide impregnated screens in some of the forested areas. This was followed by sequential releases of gamma-sterilized male flies by light aircraft. The flies, packaged in carton release containers, were dispersed twice a week along specific flight lines separated by a distance of 1-2 km. More than 8.5 million sterile male flies were released by air from August 1994 to December 1997. A sterile to indigenous male ratio of >50:1 was obtained in mid-1995 and it increased to >100:1 by the end of 1995. As a consequence the proportion of sampled young females (1-2 ovulations), with an egg in utero in embryonic arrest or an uterus empty as a result of expulsion of a dead embryo, increased from <25% in the 1st quarter to >70% in the last quarter of 1995. In addition, the age structure of the female population became significantly distorted in favor of old flies (> or = 4 ovulations) by the end of 1995. The apparent density of the indigenous fly population declined rapidly in the last quarter of 1995, followed by a population crash in the beginning of 1996. The last trapped indigenous male and female flies were found in weeks 32 and 36, 1996, respectively. Time for 6 fly generations elapsed between the last catch of an indigenous fly and the end of the sterile male releases in December 1997.