Development of a radioimmunoassay for colony stimulating factor.

Purified L-cell colony stimulating factor (CSF) and rabbit anti-CSF serum were used to devise a radioimmunoassay for this factor. The CSF was radiolabelled with the aid of lactoperoxidase and precipitated by a double antibody technique. Addition of unlabelled CSF caused a dose-related displacement of the labelled tracer. Similar results were noted with conditioned media and murine serum. The assay required only 4 days for completion as compared with 7 days for the conventional agar gel bioassay. Moreover, the radioimmunoassay proved more sensitive and accurate than the bioassay. This technique should allow further exploration of the role of CSF in granulopoiesis.     

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Mannose 6-phosphate/insulin-like growth factor II-binding proteins in human serum and urine. Their relation to the mannose 6-phosphate/insulin-like growth factor II receptor.

Human serum and urine contain polypeptides which bind mannose 6-phosphate (M6P) and insulin-like growth factor II (IGF II) and crossreact with antibodies against the M6P/IGF II receptor. These polypeptides are considered to be fragments of the M6P/IGF II receptor. The major Mr approx. 205,000 fragment in serum and urine is about 10 kDa smaller in size than the membrane-associated receptor and is accompanied by minor forms with Mr values ranging from 104,000 to 180,000. The presence of receptor fragments in biological fluids indicates that shedding is one of the mechanisms contributing to the turnover of the M6P/IGF II receptor and that receptor fragments are part of the heterogenous group of serum proteins whic bind IGF II.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Phosphorylation and sulfation of arylsulfatase A accompanies biosynthesis of the enzyme in normal and carcinoma cell lines

Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.     

Release from mouse macrophages of acidic isoferritins that suppress hematopoietic progenitor cells is induced by purified L cell colony stimulating factor and suppressed by human lactoferrin

Purified mouse L cell colony-stimulating factor (CSF) and purified iron-saturated human lactoferrin (LF) were assessed for their effects on release of acidic isoferritin-inhibitory activity (AIFIA) from resident peritoneal and spleen macrophages of B6D2F1 mice. Constitutive release of AIFIA was dependent on the number of macrophages conditioning the culture medium. Detection of release of AIFIA required at least 10(4) macrophages/ml, and increased release was noted with increased concentrations of cells. This release was enhanced by CSF and was induced by CSF from concentrations of 10(3) macrophages/ml, from which constitutive release of AIFIA was not detected. Increased concentrations of CSF induced increased release of AIFIA. The inducing effect was removed by pretreating CSF with rabbit anti-L cell CSF serum. LF suppressed the constitutive as well as the CSF-induced release of AIFIA, but results were dependent on the relative concentrations of LF and CSF used. The suppressive effects of LF were removed by pretreating LF with goat anti-human LF. Constitutive, but not CSF-induced, release of AIFIA could be ablated by removal of Ia antigen-positive macrophages with low concentrations of monoclonal anti-Ia plus complement. Treating macrophages with higher concentrations of anti-Ia in the absence of complement blocked the LF suppression of constitutive AIFIA release but not the CSF-induction of AIFIA release. Release of AIFIA from mouse macrophages can be modulated by CSF and LF. This modulation may be of significance for the regulation of myelopoiesis.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

An in silico analysis on the photoproteins Mnemiopsin 1 and Mnemiopsin 2 to explain the experimental results

Mnemiopsin 1 (Mn1) and Mnemiopsin 2 (Mn2) are photoproteins found in Mnemiopsis leidyi. We have tried to answer the question of whether the structural features of photoproteins can explain the observed activity data. According to the activity measurements data, they have the same characteristic wavelength. However, the initial intensity of Mn2 is significantly higher than that of Mn1, and decay time of Mn1 (0.92 s⁻¹) is lower than that of Mn2 (1.46 s⁻¹). The phylogenetic analysis demonstrates that, compared with Obelin and Aequorin from Obelia longissima and Aequorea victoria, respectively, a gene modification event may have caused the expansion of the N-terminal side of all photoproteins from M. leidyi. An in silico study has shown that the stability of the photoprotein–substrate complex of Mn2 is higher than that of Mn1, indicating a higher affinity of the substrate for Mn2 compared with Mn1. It was revealed that the active EF-hand loops 1 and III in Mn2 is locally more rigid compared with those in Mn1. We concluded that different stability of the photoprotein complexes leads to different initial intensity. While different patterns of the local dynamics of loops I and III may influence the decay rate.     

Fullerenol [60] Nano-cages for Protection of Crops Against Oxidative Stress: A Critical Review

Journal of Plant Growth Regulation - Fullerenols are carbon nanoparticles that have been declared as free radical sponges. There is a need to take a prudent path toward its applications in various...     

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.