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Biomedical subjects

G Rutter

Publications and source records attributed to G Rutter.

At least 19 recordsLinked to original sources
PubMed2001

The Mus cervicolor MuLV isolate M813 is highly fusogenic and induces a T-cell lymphoma associated with large multinucleated cells.

M813 is a type-C murine leukemia virus (MuLV) isolated from the Asian rodent Mus cervicolor. We have recently demonstrated that M813 defines a distinct MuLV receptor interference group. Here we show that M813 rapidly induces fusion of MuLV-expressing fibroblasts from "without," with syncytia being observed within 1 h after exposure to virus. Infection of fibroblasts with MuLV from all tested receptor-interference groups imparts susceptibility to M813-induced fusion, provided the cells also express the M813 receptor. Syncytium induction is also observed in vivo; mice infected with M813 develop a peripheral T-cell lymphoma, which is associated with large multinucleated cells of macrophage origin. A recombinant Moloney MuLV/M813 chimeric virus demonstrated that syncytium induction is a function of the Env SU protein. We postulate that the highly fusogenic property of M813 is attributable to either its unique receptor usage or sequences in the proline-rich domain of the Env protein.

Animals↗
PubMed2001

Characterization of cell lines carrying self-replicating hepatitis C virus RNAs.

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. Körner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (t(1/2)s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t(1/2), approximately 7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, replicon-bearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.

Hepacivirus↗
PubMed2001

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

3T3 Cells↗
PubMed2001

Study of the regulation by nutrients of the expression of genes involved in lipogenesis and obesity in humans and animals.

Dietary digestible carbohydrates are able to modulate lipogenesis, by modifying the expression of genes coding for key lipogenic enzymes, like fatty acid synthase. The overall objective of the Nutrigene project (FAIR-CT97-3011) was to study the efficiency of various carbohydrates to modulate the lipogenic capacity and relevant gene expression in rat and human species (control and obese subjects) and to understand the underlying molecular mechanisms involved in the regulation of lipogenic genes by carbohydrates. Key cellular mediators (namely SREBP-1c and 2, AMP activated protein kinase, cholesterol content) of the regulation of lipogenic gene expression by glucose and/or insulin were identified and constitute new putative targets in the development of plurimetabolic syndrome associated with obesity. In humans, hepatic lipogenesis and triglyceride synthesis, assessed in vivo by the use of stable isotopes, was promoted by a high-carbohydrate diet in non obese subjects, and in non alcoholic steatotic patients, but was not modified in the adipose tissue of obese subjects. Non digestible/fermentable carbohydrates, such as fructans, were shown to decrease hepatic lipogenesis in non obese rats, and to lessen hepatic steatosis and body weight in obese Zucker rats. If confirmed in obese humans, this would allow the development of functional food able to counteract the metabolic disturbances linked to obesity.

Adipose Tissue↗
PubMed2000

CDw60: an antigen expressed in many normal tissues and in some tumours.

CDw60 is a recently described T-cell antigen, which functionally delivers a costimulatory signal in T-cell activation. In addition, CDw60 has been regarded as a melanoma-associated antigen. To date, only limited information exists on the distribution of CDw60 in other normal and pathologically altered tissues in human. In the present study, the expression of CDw60 was analysed immunohistologically in a large panel of formalin-fixed and paraffin-embedded normal and pathological human tissues. The antigen was detected in several normal tissues, such as epithelia of the reproductive system, exocrine and endocrine glands, glial cells and neurons of the central and peripheral nervous systems, and lymphoid cells. These showed different subcellular distribution patterns, i.e. (1) cell surface labelling of peripheral lymphocytes and lymphocytes of the lymph node and thymus, (2) diffuse cytosolic staining in lymphocytes, subpial glial processes, and the outer plexiform layer of the retina, (3) granular cytoplasmic staining associated with the Golgi apparatus in epithelial cells of certain endocrine and exocrine glands, of the ductus epididymis and deferens, neurons of the peripheral and central nervous system, and lymphocytes and megakaryocytes of the bone marrow. In exocrine glands, e.g. of the prostate and uterine corpus, CDw60-positive Golgi fields were located in the juxtaluminal cell compartment, thus reflecting a polarized distribution. In some malignant tumours, the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were disorderly distributed throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Only in mammary carcinomas was abnormal cell surface labelling detected. A putative de novo expression of CDw60 was observed in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland, seminoma, embryonal and teratocarcinoma of the testis, small cell carcinoma of the lung, and malignant melanoma. These results define the CDw60 determinant as a broadly distributed antigen within a large panel of normal human tissues. The antigen is also detectable in some previously undescribed benign and malignant tumours, which may give importance to CDw60 as a possible diagnostic marker.

Animals↗
PubMed2000

Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus.

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.

3T3 Cells↗
PubMed2000

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV.

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

Actins↗
PubMed2000

A block to human immunodeficiency virus type 1 assembly in murine cells.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.

3T3 Cells↗
PubMed1999

Primary structure of the antigen-binding domains of a human oligodendrocyte-reactive IgM monoclonal antibody derived from a patient with multiple sclerosis.

Several murine IgM monoclonal antibodies (mAbs) promoting remyelination in mice were shown to be germline gene-encoded natural autoantibodies that react with oligodendrocytes and intracellular antigens. Here, we show that human oligodendrocyte-reactive IgM mAb DS1F8 derived from a patient with multiple sclerosis targets microtubule-like structures similar to the murine mAbs. Sequencing of the cDNAs of the variable regions revealed that the antigen-binding domains are also encoded by germline genes. These similarities of mAb DS1F8 to the murine mAbs promoting remyelination suggest that this human mAb is a natural autoantibody. This may imply that the engineering of human autoantibodies for therapy of demyelinating diseases is feasible.

Amino Acid Sequence↗
PubMed1999

Cyclophilin A incorporation is not required for human immunodeficiency virus type 1 particle maturation and does not destabilize the mature capsid.

The cellular protein cyclophilin A (CypA) is packaged into human immunodeficiency virus type 1 (HIV-1) virions through a specific interaction with the capsid (CA) domain of the Gag polyprotein. CypA is important for infectivity, but its role in viral replication is currently unknown. Previous reports suggested that CypA promotes uncoating or enhances maturation. We analyzed the morphology and capsid stability of HIV-1 variants defective in CypA binding and of virus grown in the presence of cyclosporin. Both cyclosporin treatment and alteration of Gly89 or Pro90 in the CypA-binding site of CA caused a 5- to 20-fold decrease in CypA incorporation. Virus produced from cyclosporin-treated cells and variants G89V and G89A were 10- to 100-fold less infectious but exhibited normal virion morphologies with regular cone-shaped capsids. Irregular capsid morphologies and lower infectivities were observed for some other variants in the CypA-binding region. Decreased CypA incorporation did not reduce the kinetics of intracellular polyprotein processing or of virus release. No increase in immature particles was observed. These results suggest that CypA does not promote virion maturation. Furthermore, detergent stripping of virus particles with various CypA contents revealed no difference in capsid stability. Based on these results and those reported in the accompanying paper, it appears likely that CypA also is not an uncoating factor. Alternative models for CypA function are discussed.

Capsid↗
PubMed1998

High-pressure freezing and freeze-substitution of native rat brain: suitability for preservation and immunoelectron microscopic localization of myelin glycolipids.

Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R-mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this "postembedding glycolipid-labeling technique" holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level.

Animals↗
PubMed1998

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast.

Expression of gangliosides is affected in various ways by malignant cell transformation. In the present study, we investigated the expression of CDw60, a constituent of O-acetylated disialogangliosides, in benign and atypical proliferative breast diseases, and preinvasive and invasive carcinomas by immunohistochemistry and thin-layer chromatography (TLC). In normal ducts, antibodies to CDw60 (mAb M-T21) reacted to membranes of the Golgi apparatus in the juxtaluminal cell compartment. A similar polarized distribution of Golgi cisterns in epithelial cells was observed in several benign lesions, i.e., fibroadenomas, intraductal papillomas, and gynecomastia. In contrast, blunt duct adenosis and duct hyperplasia exhibited an abnormal cytosolic and cell surface staining, whereas atypical duct hyperplasia showed randomly dispersed immunoreactive Golgi cisterns, indicating loss of epithelial polarity. In mammary carcinomas and in two breast carcinoma cell lines (MCF-7 and EFM-19) the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were distributed in a disorderly fashion throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Additionally, only well differentiated ductal carcinomas in situ or invasive ductal carcinomas disclosed a strong cell surface labelling, which was absent in lower differentiated carcinomas of the same types. In all carcinomas, the intensity of CDw60 immunostaining decreased with progressing loss of differentiation (grade of dedifferentiation), as demonstrated by staining intensity in paraffin sections and by evaluation of the relative amounts of extracted 9-O-acetyl GD3 by TLC. Our results indicate that abnormal CDw60 expression is already detectable in benign proliferative breast lesions with different risk rates to develop into malignant lesions. Downregulation of CDw60 expression in poorly differentiated invasive carcinomas may be the consequence of loss of cell functions usually associated with poor prognosis.

Adult↗
PubMed1998

Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites.

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

Amino Acid Sequence↗
PubMed1996

Detection and spatial distribution of IL-2 receptors on mouse T-lymphocytes by immunogold-labeled ligands.

To identify the plasma membrane (PM) structures implicated in T-cell activation, we studied the distribution of interleukin-2 receptors (IL-2R) and the surface topography of lymphocytes by affinity labeling in electron microscopy (EM). In particular, we analyzed the distribution of the IL-2R alpha-chain on CTLL-2 cells (a murine cytotoxic T-cell lymphoma line). Some of our experiments were extended to the functionally and morphologically distinct cell line EL4 (a routine helper T-cell lymphoma line). As affinity ligands we used a rat monoclonal antibody (clone 7D4) reactive with the routine alpha-chain of IL-2R and recombinant mouse IL-2 (rIL-2). The distribution of IL-2R was visualized on the cell surface by ligands coupled to colloidal gold particles of different sizes. Unfixed cells were labeled with gold probes and attached to concanavalin A (ConA)-pretreated coverslips. Subsequently, the cells were prepared for EM. Examination of ultrathin sections and large surface replicas revealed a high degree of variability in cell morphology and in the density of the randomly distributed gold-labeled ligands among CTLL cells. According to their typical appearance, lymphocytes with strong receptor expression can be easily identified within the cell population. In contrast, the label on many mitogen-activated EL4 cells showed a cap-like polar distribution. The results suggest the existence of diverse distribution patterns of IL-2R on CTLL and EL4 cells. These differences are believed to reflect the different physiological roles played by T-cell subsets in the immune system.

Animals↗
PubMed1995

A sulfatide-reactive human monoclonal antibody obtained from a multiple sclerosis patient selectively binds to the surface of oligodendrocytes.

In a previous paper, we described the production of a sulfatide-reactive IgM antibody-secreting B cell line that was obtained by Epstein-Barr virus transformation of peripheral B cells from a patient with multiple sclerosis (MS) (Uhlig and Dernick, 1989). In the present study, we demonstrate that this human monoclonal antibody (humAb) DS1F8 selectively binds to the surface of living oligodendrocytes in mixed brain cell cultures of newborn rats. Since a mouse mAb reactive with sulfatide was shown to inhibit oligodendrocyte progenitor differentiation, autoantibodies with binding specificities similar to DS1F8 could play a role in the demyelinating process in the CNS.

Animals↗
PubMed1993

Label-fracture of plasma membranes isolated from measles virus-infected cells.

We present a method that permits correlation of the intramembrane architecture of plasma membrane fracture faces with the distribution of specific molecules at the corresponding cytoplasmic or exoplasmic membrane surfaces. HeLa cells infected with measles virus were used as a model system. Large fragments of the dorsal membrane were isolated after the virus glycoproteins were tagged at the outer cell surface with immune serum and protein A-gold markers. In a second step, different virus polypeptides at the inner cell surface were also identified by a smaller gold label. Thereafter, the isolated plasma membranes were frozen and freeze-fractured. The complementary fracture faces were shadowed with heavy metals and carbon and examined in the transmission electron microscope without cleaning of remaining biological material. Thus, the micromorphology of the replicated fracture faces and the topochemistry of virus components localized at the corresponding leaflets of the plasmalemma could be seen on the same image at high resolution. Of note is that the freeze-fracture morphology of the protoplasmic face is related to the molecular composition of the cytoplasmic surface, as revealed by antibody tagging.

Cell Membrane↗