The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae. Isolation of mutants and cloning of the EPT1 gene

A colony autoradiographic assay was used to identify nine Saccharomyces cerevisiae mutants defective in in situ ethanolaminephosphotransferase activity (ept mutants). Genetic analysis revealed five complementation groups. The EPT1 gene was cloned by complementation of ept1 using a yeast genomic library and was localized to a 2.1-kilobase region of DNA. An ept1 deletional mutant was constructed and introduced into the chromosome by integrative transformation. The ethanolaminephosphotransferase activities in membranes prepared from ept1 and ept2 mutants were reduced 30- to 90-fold and 2- to 3-fold compared with wild-type activity, respectively; the other ept mutants had activities similar to wild type. In strains transformed with a multicopy EPT1-bearing plasmid, a 22- to 33-fold overproduction of ethanolaminephosphotransferase activity was observed. The sn-1,2-diacylglycerol cholinephosphotransferase activities in membranes prepared from ept1 mutants were reduced 3.5- to 7-fold. In contrast to the residual CMP-sensitive cholinephosphotransferase activity observed in cpt1 mutants (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), the residual cholinephosphotransferase activity of ept1 mutants was CMP-insensitive. The cholinephosphotransferase activities in strains bearing the EPT1 gene on multicopy plasmids were elevated 13- to 23-fold and were CMP-sensitive. The data indicate that 1) the cloned EPT1 gene most likely represents the structural gene for the yeast ethanolaminephosphotransferase, 2) the EPT1 gene product possesses both ethanolamine- and cholinephosphotransferase activities, and 3) the EPT1 gene is nonessential for growth.     

An islet amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing

Amyloid deposits occurring in the islets of Langerhans in patients with noninsulin-dependent diabetes mellitus and some insulinomas contain a 37-amino acid peptide that is structurally related to calcitonin gene-related peptide. We have identified three cDNA clones encoding islet amyloid polypeptide (IAPP) or diabetes-associated peptide (DAP) by oligonucleotide screening of a lambda gt10 human insulinoma cDNA library. Two of the three cDNAs contained a domain encoding IAPP/DAP but had an intron-like sequence in their 5' region. The other cDNA contained an open reading frame encoding an 89-amino acid precursor having a typical signal peptide followed by a small prohormone-like sequence containing within it the IAPP/DAP peptide bracketed at its NH2 and COOH termini by Lys-Arg and Gly-Lys-Arg, respectively. These data indicate that this amyloid peptide is generated by proteolytic processing similar to that for proinsulin and other islet prohormones and also that the peptide may be carboxyamidated. The isolation of cDNA clones having 5'-unprocessed intron-like sequences suggests that inefficient or alternative splicing of this mRNA occurred in the insulinoma.     

The microheterogeneity of androgen-binding protein in rat serum and epididymis is due to differences in glycosylation of their subunits

The microheterogeneity of androgen-binding protein (ABP) from rat serum and epididymis was examined by subjecting purified native or deglycosylated preparations to analysis by one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to nitrocellulose and immunochemical localization. Analysis of native ABP by one-dimensional sodium dodecyl sulfate-PAGE confirmed earlier observations that it is composed of subunits and that the subunits of serum ABP had higher apparent molecular weights than those of epididymal ABP. Treatment with neuraminidase, N-glycanase, or O-glycanase, alone or in combination, resulted in decreases in the apparent molecular weight of the subunits. These analyses indicated that terminal sialic acid residues and Asn-linked oligosaccharides were present on both subunits of ABP from the two sources. The fact that the greatest reduction in the Mr of the heavy subunit occurred following treatment with all three enzymes provides evidence that O-linked sugars are present on it. While enzyme treatment did not result in the appearance of a single subunit, chemical deglycosylation did (Mr 39,600). The carbohydrate composition of the heavy and light subunits of intact serum and epididymal ABP was 22 and 9% and 19 and 8%, respectively. Analysis by two-dimensional PAGE indicated that both subunits of the ABPs were composed of isoelectric variants. Although ABP from the two sources had several variants in common, differences were also observed. Treatment of the ABPs with the enzymes resulted in a shift of the pI values to a more basic pH range, indicating that carbohydrate removal also removed charged moieties. The most dramatic shift in the pI values of the isoforms occurred when O-glycanase was present in the enzyme mixture, providing further evidence for the presence of O-linked oligosaccharides on ABP. Isoelectric variants were present even after chemical deglycosylation of ABP.     

M?ssbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe M?ssbauer spectroscopy. The M?ssbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.     

Histidyl-tRNA synthetase, the myositis Jo-1 antigen, is cytoplasmic and unassociated with the cytoskeletal framework

The myositis-specific anti-Jo-1 autoantibody, which is directed against histidyl-tRNA-synthetase, is found in 30% of polymyositis patients. The Jo-1 antigen has been reported to be a nuclear antigen by some authors. On the contrary we show that less than 2% of the total histidyl-tRNA and lysyl-tRNA synthetase activities are associated with purified rat liver nuclei or the hepatocyte intermediate filament-nuclear fraction. In the presence of polyethylene glycol, in which the high Mr multi-enzyme complex containing lysyl-tRNA synthetase is insoluble, 65% of the lysyl-tRNA synthetase and only 15% of histidyl-tRNA synthetase activities remained associated with the cytoskeletal framework. The Jo-1 antigen exhibited a diffuse granular cytoplasmic distribution in cultured rat hepatocytes as determined by indirect immunofluorescent microscopy. Hence, the Jo-1 antigen is cytoplasmic and unassociated with the cytoskeletal framework or high Mr synthetase complex in situ.     

Asbestos bodies in bronchoalveolar lavage fluid. A study of 20 asbestos-exposed individuals and comparison to patients with other chronic interstitial lung diseases

We studied the asbestos body (AB) content of bronchoalveolar lavage fluid from 20 patients with a history of occupational asbestos exposure, 31 patients with sarcoidosis and 5 patients with idiopathic pulmonary fibrosis. The cellular lavage pellet was digested in sodium hypochlorite and filtered onto Nuclepore filters for AB quantification by light microscopy. ABs were found in 15 of 20 asbestos-exposed individuals, 9 of 31 sarcoidosis cases and 2 of 5 patients with idiopathic pulmonary fibrosis. There was a statistically significant difference in the number of ABs per million cells recovered or per milliliter of recovered lavage fluid in the asbestos-exposed group as compared to the other categories of chronic interstitial lung disease. The highest levels occurred in patients with asbestosis. Large numbers of asbestos bodies in the lavage fluid (greater than 1 AB/10(6) cells) were indicative of considerable occupational asbestos exposure, whereas occasional bodies were a nonspecific finding.     

Relationships between the human pepsinogen DNA and protein polymorphisms.

Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. The PGA gene family exhibits polymorphic variation in human populations that can either be demonstrated by electrophoretic analysis of the proteins or by analysis of the respective genes with cDNA probes. Here, we describe the interrelationships between the most common pepsinogen protein phenotypes and the corresponding pepsinogen haplotypes (A, B, and C) containing different combinations of the PGA3, PGA4, and PGA5 genes. We propose that this unusual genetic variation involving haplotypes that contain three, two, and one genes, respectively, is the result of molecular evolution by gene duplication.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Contribution of macrophages to immediate hypersensitivity reaction

The interaction of mast cells with other leukocytes during immediate hypersensitivity reactions was tested by in vivo and in vitro experiments. Intraperitoneal challenge of passively sensitized rats with antigen caused the production of peptidoleukotrienes, leukotriene (LT)B4, thromboxane (TX)B2, and 6-keto-prostaglandin (PG) F1 alpha in the peritoneal cavity. Pretreatment of the rats with thioglycollate i.p. markedly changed the amount of eicosanoids formed. When polymorphonuclear leukocytes were the predominant cell type in the peritoneal exudate, both LTC4 and 6-keto-PGF1 alpha were decreased by 75% each and TXB2 by 50%. When elicited macrophages were predominant, there was an additional reduction in LTC4 by 68% as compared with 18 hr after thioglycollate treatment, but no additional change in the other arachidonic acid metabolites. In vitro antigen challenge of passively sensitized mouse bone marrow-derived mast cells caused the release of LTC4, LTB4, 6-trans-LTB4, 5-hydroxyeicosatetraenoic (5-HETE), and TXB2. Exposure to antigen of these mast cells in the presence of resident peritoneal macrophages markedly altered eicosanoid formation. Early in the time course (2 to 15 min), macrophages markedly enhanced all 5-lipoxygenase products. However, later in the time course (30 to 120 min), these products were decreased. This decrease was reversed by catalase and superoxide dismutase, which suggests the involvement of oxygen radicals. These active oxygen species also seemed to be generated by mast cells, because these enzymes caused an increase in 5-lipoxygenase products when mast cells were challenged alone. RIA of cyclooxygenase products showed that mast cells released only TXB2 when stimulated with antigen. When they were stimulated in the presence of macrophages, TXB2 and also PGE2 and 6-keto-PGF1 alpha were synthesized. Therefore, macrophages probably contribute the PGE2 and 6-keto-PGF1 alpha. Because the same amount of TXB2 was generated whether macrophages were present or not, the mast cells seem to be the major source of this compound. These data indicate that macrophages and possibly polymorphonuclear leukocytes participate in immediate hypersensitivity reactions.