Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Chromosomal localization of ARSB,the gene for human N-acetylgalactosamine-4-sulphatase

Summary A deficiency of N-acetylgalactosamine-4-sulphatase (G4S, gene symbol ARSB), results in the accumulation of undegraded substrate and the lysosomal storage disorder, Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). In situ hybridization using an ³H-labelled human G4S genomic DNA fragment to human metaphase chromosomes localized ARSB to chromosome 5q13–5q14. This location is consistent with, an refines, previous chromosomal assignments based on the expression of human G4S in somatic cell hybrids.     

Non-photochemical quenching of Fo in leaves is emission wavelength dependent: consequences for quenching analysis and its interpretation

The effects of light-induced non-photochemical quenching on the minimal F_o, and variable F_v, fluorescence emissions at 690 and 730 nm in leaves were determined. Non-photochemical quenching of F_o, but not F_v, was found to be dependent upon the wavelength of emission, and was greater at 690 nm than at 730 nm. For emission at 730, compared to at 690 nm, approx. 30% of F_o was not affected by non-photochemical quenching processes in leaves of C3 plants; in maize leaves this was found to be approx. 50%. The data indicate that a substantial proportion of the pigments contributing to F_o emission at 730 nm are not quenched by light-induced, non-photochemical quenching processes and that there are large differences in the pigment matrices contributing to F_o and F_v emissions at 730 nm, compared to those at 690 nm. These findings have important implications for the accurate estimation and interpretation of non-photochemical quenching of fluorescence parameters and their use in the calculation of photochemical efficiencies in leaves. Measurements of fluorescence emissions at wavelengths above 700 nm are likely to give rise to significant errors when used for determinations of photochemical and non-photochemical quenching parameters.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides.

Cells of the oral bacterium Streptococcus gordonii express three cytoplasmic membrane-bound lipoproteins with apparent molecular masses of 76 to 78 kDa that are the products of three genes (designated hppA, hppG, and hppH). The lipoproteins are immunologically cross-reactive, contain 60% or more identical amino acid residues, and are highly similar to the AmiA, AliA (PlpA), and AliB substrate-binding protein components of an oligopeptide permease in Streptococcus pneumoniae. Insertional inactivation of the hppA or hppH gene resulted in loss of the ability of S. gordonii cells to utilize specific peptides of five to seven amino acid residues for growth. An insertion within the COOH-terminal coding region of hppG that caused apparent truncation of the HppG polypeptide had a similar effect; however, S. gordonii mutants in which HppG polypeptide production was abolished were still able to grow on all oligopeptides tested. Inactivation of hppA gene (but not inactivation of the hppG or hppH gene) caused reduced growth rate of cells in complex medium, slowed the rate of development of competence for transformation, reduced the efficiency of transformation, and increased the resistance of cells to aminopterin. These results suggest that the formation of a solute-binding-protein complex consisting of at least the HppA and the HppH lipopolypeptides is necessary for binding and subsequent uptake of primarily hexa- or heptapeptides by a Hpp (Hexa-heptapeptide permease) system in S. gordonii. In addition, Hpp may play a role in the control of metabolic functions associated with the growth of streptococcal cells on complex nitrogen sources and with the development of competence.