Vacuolar cytoplasmic phase separation in cultured mammalian cells involves the microfilament network and reduces motional properties of intracellular water

Hep-2, human epithelial carcinoma cells, and human foreskin fibroblasts (FF9 and FF13) were exposed to either an ultrafiltrate (< 50 kD) of human sera or the weak base, procaine hydrochloride, to induce reversible cytoplasmic vacuolization. The formation of vacuoles was shown not to be due to imbibition of medium. Ultrastructural details obtained from various stages of vacuole formation were compared. In both cases of induction vacuoles were irregular and often appeared membraneless, with little in the way of electron-dense content. They started to form in the perinuclear cytoplasm and progressed towards the periphery. Osmotic stress was not involved since mitochondria remained normal throughout a vacuolization episode.
Vacuoles were often seen in close contact with filamentous structures, and this association remained detectable at late stages of the phenomenon. Fluorescent visualization of F-actin confirmed that the vacuoles were frequently bordered by microfilaments. No major metabolic impairment was apparent in vacuolized cells as judged by protein synthesis measurements, but nuclear fluorescence (DNA content) and forward light scatter (nuclear volume) by flow cytometric analysis suggested late S phase and G2 retardation. ¹H-nmr relaxation measurements indicated intracellular water restricted in motional characteristics in vacuolized cells. The possibility of a restricted cytoplasmic phase separation as part of a transient adaptation response is raised, and a hypothesis to explain the findings is discussed.     

Regulation of neuronal differentiation in neuron-enriched primary cultures from embryonic rat cerebra by platelet activating factor and the structurally related glycerol ether lipid, dodecylglycerol.

Primary cultures enriched in neurons dissociated from embryonic rat cerebra were used to demonstrate that platelet activating factor and the structurally related ether glycerolipid, dodecylglycerol, are readily taken up in small amounts by neurons and that they stimulate the differentiation of neurons. The stimulation of neuronal differentiation was observed as a precocious development of axon-like extensions which correlated with a concentration-dependent increase in neuronal-specific enzyme activities. This stimulation of morphological and neurochemical factors by either platelet activating factor or dodecylglycerol was almost completely abolished by triazolam, a known inhibitor of platelet activating factor function. Neither platelet activating factor nor dodecylglycerol at the concentrations used to achieve stimulation of neuronal differentiation compromised the plasma membrane, as indicated by the lack of leakage of cytoplasmic lactic acid dehydrogenase.     

Study on lymphocyte activation and proliferation induced by anti-CD3 McAb

Summary T cell activation and proliferation via CD₃-TCR complex were investigated by lymphocyte DNA synthesis in vitro. Several interfering factors were also discussed. The result indicated that lymphocyte activation and proliferation are calciumdependent. A rise of cytoplasmic free Ca²⁺ quickly following activation with CD₃ McAb is mainly due to intracellular mobilization of Ca²⁺, while lymphocyte proliferation needs both intracellular mobilization of Ca²⁺ as well as influx of extracellular Ca²⁺. It was confirmed that CTX sensitive G protein plays a role in regulating T cell proliferation by pretreatment with CTX suppressing lymphocyte³H-TdR incorporation obviously. PLC and PKC inhibitor neomycin and P. S. S could also decrease T cell proliferation.     

Antineutrophil cytoplasmic antibodies in IgA nephropathy and Henoch-Sch?nlein purpura.

The frequency, isotype, and specificity of antineutrophil cytoplasmic antibodies were investigated in a cross-sectional study of 100 patients with IgA nephropathy and 30 children with Henoch-Sch?nlein purpura. Two of the patients with IgA nephropathy had high titres of antineutrophil cytoplasmic antibodies which were of IgG isotype and confirmed as antimyeloperoxidase antibodies in solid-phase ELISA and inhibition experiments. Antineutrophil cytoplasmic antibodies were not detected in the children with Henoch-Sch?nlein purpura and none of the patients in either group had IgA antineutrophil cytoplasmic antibodies. A further 20 IgA nephropathy and 10 Henoch-Sch?nlein purpura patients were studied longitudinally in different clinical phases at 4-monthly intervals over a 2-year period. None of these patients had or developed antineutrophil cytoplasmic antibodies. We conclude that IgA antineutrophil cytoplasmic antibodies are not involved in the vasculitis of Henoch-Sch?nlein purpura or in the pathogenesis of glomerular injury in IgA nephropathy. The detection of IgG antimyeloperoxidase antibodies in a small minority of IgA nephropathy patients extends the spectrum of diseases associated with autoimmunity to this antigen but is of uncertain significance.     

Antineutrophil cytoplasmic antibodies (ANCA) in idiopathic pulmonary hemosiderosis

Four children were diagnosed with idiopathic pulmonary hemosiderosis (IPH), over a period of 4 years. Retrospectively, antineutrophil cytoplasmic antibodies (ANCA) were studied by indirect immunofluorescence (IIF) and ELISA in 18 sera from these patients, stored at -20°C. ANCA-positive sera, from 1/20 to 1/1, 200 dilution, were found in 3/4 of the patients, by IIF. The patient with the highest titre of ANCA died 3 months later during an acute crisis, the other two patients need a minimal dose of steroids. In one case only, a patient who is still without treatment, had no ANCA. The antibodies anti-myeloperoxidase and anti-pro-teinase-3 were negative or at border line levels. Rheumatoid factor, anti-nuclear (Hep-2), anti-endomysial, anti-reticulin and antibasement membrane antibodies were negative in all sera. The surviving patients were followed-up for more than 10 years with no systemic or renal disease appearences. The presence of serum ANCA may help to classify children with pulmonary haemorrhage and may have a prognostic value.     

Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

Identification of a Thymidylate Synthase Gene within the Genome of Chilo Iridescent Virus

The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.     

Effects of epidermal growth factor in the final stages of nuclear and cytoplasmic oocyte maturation in humans

Evidence has accumulated in mammals suggesting a positive rolefor epidermal growth factor (EGF) as an inducer of oocyte maturation.The potential use of EGF as inducer of cytoplasmic and nuclearmaturation was tested in women with > 10 oocytes retrievedin in-vitro fertilization (IVF), since we have previously observedthat such oocytes are immature. Oocytes from 17 high responderswere randomly allocated to one of the three treatment groupsupon retrieval: control receiving no EGF (n = 93), 1.0 ng/mlEGF (n = 92) and 10.0 ng/ml EGF (n = 77) for 6 h before insemination.The rates of fertilization were respectively 54.6, 59.0, and46.1%, suggesting that EGF is not effective at this maturationalstage after this length of exposure. Embryo development wasfurther analysed by the appearance of the embryos under thedissecting microscope and the number of blastomeres developed48 h after insemination. No difference between groups was observedconsidering the number of blastomeres developed. However, embryosderived from oocytes treated with 10 ng/ml EGF displayed a worseappearance under the microscope. It is concluded that a 6 hincubation with EGF does not seem to affect cytoplasmic maturationin oocytes obtained after gonadotrophin treatment, as ascertainedby the rate of fertilization following oocyte insemination.