Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.     

Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture.

Elevated fibronectin (Fn) and Fn fragment concentrations are found in the synovial fluid of osteoarthritic and rheumatoid arthritic patients. Fn has been shown to affect expression of chondrocytic matrix proteins, and Fn fragments have been shown to elevate gene expression of neutral proteinases in synoviocytes. For these reasons, we tested the effects of Fn fragments on protease release and resultant proteoglycan release from cartilage in serum-free bovine articular cartilage explant cultures. We have found that 1 microM amino-terminal 29- and 50-kDa gelatin-binding Fn fragments caused over a 50-fold enhancement of gelatinolytic and collagenolytic proteinase release with a 23-fold enhancement of proteoglycan (PG) release. Release was significant at fragment concentrations as low as 20 nM. An integrin-binding 140-kDa fragment mixture was the least active fragment, whereas native Fn had little activity. The relative activities of the fragments correlated with their relative abilities to bind to cartilage. The RGDS integrin-recognition peptide also caused release, although sequence mutants did not. PG release was blocked by actinomycin D, cycloheximide, and deoxyglucose. Fn fragment-mediated PG release was decreased in 10% serum by over 10-fold but was still 2-fold greater than in controls. In the presence of insulin-like growth factor-1, PG release was as great as without serum. We suggest that Fn fragments, as found in diseased synovial fluid, may contribute to protease-mediated damage to cartilage.     

Comparative response of nestling European starlings and red-winged blackbirds to an oral administration of either dimethoate or chlorpyrifos.

Red-winged blackbird (Agelaius phoeniceus; blackbird) and European starling (Sturnus vulgaris; starling) nestlings were dosed with either 2.0 mg/kg body mass chlorpyrifos, 50.0 mg/kg body mass dimethoate, or a propylene glycol carrier in situ. Four growth measurements (body mass, culmen, tarsus, wing) were recorded from nestlings to determine if these organophosphorus compounds caused perturbations in development at sublethal concentrations. Blackbird nestlings were more sensitive to chlorpyrifos than starling nestlings were more sensitive to dimethoate than blackbird nestlings. This was in contrast to reported adult LD50 values where the reverse was true. Blackbird nestlings were more tolerant of a substantially higher concentration of dimethoate than the adult LD50. The sensitivity of starling nestlings to dimethoate was similar to adults. In contrast, juveniles of both species were more sensitive to chlorpyrifos than adults. After the initial 24 hr, surviving nestlings dosed with either chemical recovered and continued their development. Exposure to dimethoate caused significant depression in starling body mass during the initial 24 hr period. Survivors obtain body mass equal to controls within 48 hr post dosing. The research presented here demonstrates that the simple supposition that passerine nestlings are typically more sensitive to toxins than adults does not always hold true. It also indicates that sensitivity relationships among adults do not necessarily apply to their nestlings.     

Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.     

The Marfan syndrome locus: confirmation of assignment to chromosome 15 and identification of tightly linked markers at 15q15-q21.3

The Marfan syndrome is a common autosomal dominant disorder of connective tissue. Despite many years of intensive investigation, the primary genetic defect has not yet been identified. Reverse genetic methods, targeted at mapping this disease gene, have resulted in an initial report of linkage of the genetic locus for the Marfan phenotype in Finnish families to two polymorphic markers on chromosome 15. We have investigated four large multiplex American families with classic Marfan syndrome using standard genetic linkage methods. Our data confirm the assignment of the Marfan syndrome gene to chromosome 15, but establish a more centromeric location (defined by markers D15S25 and D15S1) as the most probable site for the genetic defect (lod score = 12.1, theta = 0.00). These data should facilitate identification and characterization of the Marfan syndrome gene and, in selected families, have immediate application to diagnosis of equivocal cases or prenatal counseling.     

Q fever in maritime Canada.

Only nine cases of Q fever were recorded in Canada in the 20 years prior to 1978. In the 18 months from August 1979 to January 1981 the disease was diagnosed serologically in six patients from the Maritime provinces. All were epidemiologically unrelated and none had been exposed to animals. Five had pneumonia and one had chronic Q fever with probable prosthetic valve endocarditis. Three of the five pneumonia patients presented with signs and symptoms of an acute lower respiratory tract infection and were indistinguishable clinically from other patients with atypical pneumonias. The other two with pneumonia presented with nonresolving pulmonary infiltrates and complained of decreased energy. Four of the five pneumonia patients responded well to treatment with erythromycin; the fifth required two courses of tetracycline. The patient with chronic Q fever had a large amount of cryoglobulins in his serum and evidence of immune complex disease. These cases indicate that Q fever should be considered as a possible cause of atypical pneumonia in Canada.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

The effects of laminin on the growth and differentiation of embryonal carcinoma cells in defined media

In this paper we have examined the growth and differentiation of the embryonal carcinoma cell line, F₉, in the defined medium EM-3 at low density. We show that the growth of F₉ and their differentiated cells (F₉-diff) in EM-3 is strongly density dependent. At low cell densities the growth of both cell types is severely limited and most of the cells do not survive. Although this poses a problem for working with F₉ and F₉-diff in EM-3, it provides a convenient assay for identifying molecules that support their growth at low density. Using this assay, we have determined that laminin, a newly isolated glycoprotein of basement membranes, significantly improves the growth and short-term survival of both F₉ and F₉-diff. However, addition of laminin to EM-3 is insufficient to promote the clonal growth of these cell types. Our findings also indicate that laminin promotes the attachment of F₉ and F₉-diff in defined media. On the basis of our results, we propose an attachment function for laminin during the early stages of mammalian development.