Studies on a leukocyte elastase inhibitor present in the culture medium of inflamed synovial tissue

The spent medium of cultured inflamed synovial tissue contains a potent inhibitor of leukocyte elastase. This leukocyte elastase inhibitor has no effect on leukocyte cathepsin G and pancreatic elastase is only marginally affected. The inhibitor is a glycoprotein, stable to heat, acid and reductive alkylation. Pretreatment of the inhibitor with either trypsin or chymotrypsin results in its inactivation.     

Physiological and Environmental Requirements for Poplar (Populus deltoides) Bark Storage Protein Degradation

In poplar (Populus deltoides Bartr. ex Marsh), a 32-kD bark storage protein (BSP) accumulates in the bark during autumn and winter and declines during spring shoot growth. We investigated the physiological and environmental factors necessary for the degradation of poplar BSP. Poplar plants were exposed to short-day (SD) photoperiods for either 28 or 49 d. Plants exposed to short days for 28 d formed a terminal bud but were not dormant, whereas exposure to short days for 49 d induced bud dormancy. BSP accumulated in bark of plants exposed to both SD treatments. The level of BSP declined rapidly when nondormant plants were returned to long days. BSP levels did not decline in dormant plants that were exposed to long-day (LD) conditions. If dormant plants were first treated with either low temperatures (0[deg]C for 28 d) or with 0.5 M H2CN2 to overcome dormancy and then returned to long days, the level of BSP declined. Removal of buds from non-dormant or dormant plants in which dormancy had been overcome inhibited the degradation of BSP in LD conditions. BSP mRNA levels rapidly declined in plants exposed to long days, irrespective of the dormancy status of the plants or the presence or absence of buds. These results indicate that the buds of poplars are somehow able to communicate with bark storage sites and regulate poplar BSP degradation. These results further support an association of BSP mRNA levels with photoperiod because short days stimulate BSP mRNA accumulation, whereas long days result in a decline of BSP mRNA abundance.     

Concepts of hydrochemical water quality of the Delta waters

Summary Some concepts of hydrochemical water quality in estuaries are discussed from the point of view of time and space. The relations of metal and other substances with chloride as conservative property are questioned with respect to time, space and presence of particulate matter. Some examples are presented for phosphorus budgets of the Dutch Wadden Sea and Lake Grevelingen in order to demonstrate the various impacts determining estuarine conditions. The effects of non-attached particulate metals and slow solid-binding processes of metals and radionuclides onto particles have to be considered for estuarine studies. At the same time a start should be made to determine the residence times of natural and anthropogenic substances in the estuaries which reflect also the human activities on the estuaries and thus the resulting changes in the natural equilibria.Communication no. 174 of the Delta Institute for Hydrobiological Research.     

Chromosome analyses of children after ecological lead exposure

In the present work chromosome analysis was performed in a group of 30 children living in a town with a lead plant. Due to the emission of the smelter the individual lead uptake through food, drinking water and inhalation was increased. They were selected out of 1600 children whose blood lead level, delta-aminolevulinic acid dehydratase activity in the erythrocytes and erythrocyte porphyrine level was measured. In the investigated group of children the values of these parameters showed to be indicative for a significant lead exposure. A total of 10,000 cells was scored after 48 h culture time. Despite a significantly increased lead load as compared with two groups of 10 children from a suburb and the isle of Helgoland there was neither evidence for a higher number of cells with structural chromosome aberrations, nor for an increased aberration yield.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. I. Effect of size.

Aspects of protein structure determining endocytosis of proteins by sinusoidal rat liver cells in vivo have been studied, using cross-linked or aggregated derivatives of bovine pancreatic ribonuclease A (labelled with 125I) as probes. Ribonuclease was cross-linked by reaction with dimethylsuberimidate, a way of modification that does not change the charge of the protein. Monomer, dimer and polymer fractions were isolated by gel filtration and characterized in respect of size and number of amino groups modified. Maintenance of enzyme activity, stability of disulfide bonds, and lack of susceptibility to endoproteases showed that the cross-linking procedure did not result in gross conformational changes of the ribonuclease molecules. Monomer, dimer and polymer fractions were injected into nephrectomized rats and plasma clearance and uptake in liver and spleen were determined. About 30% of the injected polymer fraction was found in liver 15 min after injection; for dimer and monomer fractions values of 6% and 2% of the dose were found. Similar differences were found in spleen. Autoradiography, cell isolation, and subcellular fractionation showed that in liver the radioactive proteins were taken up in lysosomes of sinusoidal cells. Similar results were obtained with fractions of aggregated ribonuclease prepared by freeze-drying the protein from 50% acetic acid. Our results demonstrate that the rate of uptake of the ribonuclease derivatives is positively correlated with the size of the molecules. Similarity of the results obtained with cross-linked and aggregated fractions suggests that the number of ribonuclease 'subunits'/molecule, rather than the procedures used to prepare the polymers, determine the rate of uptake by liver and spleen.     

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. II. Effect of charge.

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic groups on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways. Firstly, we changed the positively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolished their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polymer and 32% of the succinylated polymer were endocytosed by liver after 6 h. For the dimer fractions these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polymer in the lysosomal/microsomal fraction of sinusoidal liver cells, probably endothelial cells. Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives to the cation exchanger SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.     

Mixed-Backbone Oligonucleotides Containing Phosphorothioate and Methylphosphonate Linkages as Second Generation Antisense Oligonucleotide

Abstract

Antisense oligonucleotides are being studied as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have synthesized phosphorothioate oligonucleotides containing methylphosphonate linkages at the 5′-end, the 3′-end, or in the center, and have evaluated the impact of these linkages on the biophysical properties, biological properties, and some of the safety parameters.     

The A Allele of the rs1990760 Polymorphism in the IFIH1 Gene Is Associated with Protection for Arterial Hypertension in Type 1 Diabetic Patients and with Expression of This Gene in Human Mononuclear Cells

Background

The rs1990760 polymorphism of interferon induced with helicase C domain 1 (IFIH1) has been associated with type 1 diabetes mellitus (T1DM). Here, we investigated whether this polymorphism is associated with T1DM or its clinical characteristics in a Brazilian population, and if IFIH1 gene expression in mononuclear cells from T1DM patients differs according to the genotypes of this polymorphism. A meta-analysis was also conducted to evaluate if the rs1990760 polymorphism is associated with T1DM.

Methods

Frequencies of the rs1990760 polymorphism were analyzed in 527 T1DM patients and in 517 healthy subjects. IFIH1 gene expressions according to genotypes were measured in a sub-sample of 26 T1DM patients by quantitative real-time PCR.

Results

Our data show the association of the A allele with risk to T1DM under a dominant model of inheritance [odds ratio (OR) = 1.421, P = 0.037], adjusting for ethnicity. The meta-analysis revealed significant association between the rs199760A allele and risk for T1DM for all analyzed inheritance models. Surprisingly, T1DM patients carrying the A allele showed lower levels of systolic (P = 0.001) and diastolic (P = 1×10⁻¹⁰) blood pressures as compared to G/G carriers. Furthermore, the A/A genotype seems to be associated with protection to arterial hypertension (AH) after adjustment for covariates (OR = 0.339, P = 0.019). IFIH1 gene expression in mononuclear cells from 26 T1DM patients did not differ among genotypes (P = 0.274). Nevertheless, IFIH1 gene expression was increased in mononuclear cells from T1DM patients with AH as compared with T1DM patients without AH [6.7 (1.7–2.0) vs. 1.8 (1.3–7.1) arbitrary units; P = 0.036]. The association with blood pressures and AH was not observed in patients with type 2 diabetes mellitus.

Conclusions

Our results indicate that the rs1990760 polymorphism is associated with T1DM. Interestingly, the rs1990760 A allele seems to be associated with protection for AH in T1DM patients. Further studies are needed to confirm the association with AH.