The critical N-linked glycan of murine leukemia virus envelope protein promotes both folding of the C-terminal domains of the precursor polyprotein and stability of the postcleavage envelope complex

The infectivity of Friend ecotropic murine leukemia virus was previously shown to be highly sensitive to modification in its envelope protein (Env) at only one of the eight signals for N-linked glycan attachment, the fourth from the N terminus (gs4). In the present study, a set of six single-amino-acid substitutions in or near gs4 was used to determine the function of this region of Env and the role played by the glycan itself. One mutant that lacked the gs4 glycan was fully infectious, while one that retained this glycan was completely noninfectious, indicating that the gs4 glycan per se is not required for Env function. Infectivity correlated with the level of mature Env complex incorporated into virus particles, which was determined by the severity of defects in transport of the envelope precursor protein (gPrEnv) from the endoplasmic reticulum into the Golgi apparatus, in cleavage of gPrEnv into the two envelope subunits (the surface protein [SU] and the transmembrane protein [TM]), and in the association of SU with cellular membranes. All of the mutants induced the wild-type level of superinfection interference, indicating that the gs4 region mutations did not interfere with proper folding of the N-terminal domain of SU. These results suggest that the gs4 region mediates folding of the C-terminal domains of gPrEnv and stability of the interaction between SU and TM. Although the gs4 glycan was not essential for infectivity, processing of all mutant Envs lacking this glycan was significantly impaired, suggesting that efficient folding of gPrEnv requires a glycan at this position. The conservation of a glycosylation site homologous to gs4 across a broad range of retroviruses suggests that this sequence may play a similar role in many retroviral Envs.     

The mouse H-2A region influences the envelope gene structure of tumor-associated murine leukemia viruses

C57BL/10 (B10) strains congenic at the mouse major histocompatibility locus (H-2) were injected with a modified ecotropic SL3-3 murine leukemia virus (MuLV) to determine the effect of the H-2 genes on the envelope gene structure of recombinant MuLVs. All tested strains rapidly developed T-cell lymphomas, and recombinant proviruses were detected in the tumor DNAs by Southern blot. The B10.D2 (H-2d), B10.Br (H-2k), B10.Q (H-2q), and B10.RIII (H-2r) strains exhibited a TI phenotype in which almost all tumors contained type I recombinants. These recombinants characteristically acquire envelope gene sequences from the endogenous polytropic viruses but retain the 5' p15E (TM) gene sequences from the ecotropic virus. The parental B10 (H-2b) strain, however, had a novel phenotype that was designated NS for nonselective. Only 30% of the B10 tumors had detectable type I recombinants, whereas a proportion of the others appeared to contain type II recombinants that lacked the type I-specific ecotropic p15E gene sequences. Studies of other B10 congenic strains with hybrid H-2 loci and selected F1 animals revealed that the NS phenotype was regulated by a dominant gene(s) that mapped to the A region of H-2b. These results demonstrate that a host gene within the major histocompatibility complex can influence the genetic evolution of pathogenic retroviruses in vivo.     

Differential effects of neurotrophic factors on motoneuron retrograde labeling in a murine model of motoneuron disease

It has been shown that abnormalities in axonal transport occur in several mouse models with motoneuron degeneration and also in the human disease amyotrophic lateral sclerosis. In this report, we have examined the potential of neurotrophic factors to act on axonal transport properties in a mouse mutant, progressive motor neuronopathy (pmn). This mouse mutant has been characterized as a "dying-back" motoneuronopathy, with a loss of motoneuron cell bodies and motor fibers. Retrograde transport to the spinal cord motoneurons was determined using fluorescent tracers either injected into the gastrocnemius muscle or applied directly onto the cut sciatic nerve. Because the rate of retrograde labeling was significantly reduced in the pmn, we examined the potential of neurotrophic factors to compensate for the impairment. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) but not glial-derived neurotrophic factor (GDNF) or nerve growth factor (NGF) were capable of significantly improving the rate of labeling. The differential effects of these factors agree with previous studies showing that molecules that promote cell survival do not necessarily compensate for axonal deficiency. Because impairment of axonal properties appears as an early event in motoneuron pathology, our results may have important clinical implications in the treatment of motoneuron diseases.     

Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins

Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.     

Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus

The 10A1 murine leukemia virus (MuLV) is a recombinant type C retrovirus isolated from a mouse infected with amphotropic MuLV (A-MuLV). 10A1 and A-MuLV have 91% amino acid identity in their envelope proteins yet display different host ranges. For example, CHO-K1 cells are resistant to A-MuLV but susceptible to infection by 10A1. We have now determined that retroviral vectors bearing altered A-MuLV envelope proteins containing 10A1-derived residues at positions 71 (A71G), 74 (Q74K), and 139 (V139M) transduce CHO-K1 cells at efficiencies similar to those achieved with 10A1 enveloped vectors. A-MuLV enveloped retroviral vectors with these three 10A1 residues were also able to transduce A-MuLV-infected NIH 3T3 cells. This observation is consistent with the ability of vectors bearing this altered A-MuLV envelope protein to recognize the 10A1-specific receptor present on NIH 3T3 cells and supports the possibility that residues at positions 71, 74, and 139 of the 10A1 envelope SU protein account for the expanded host range of 10A1.     

Pushing the envelope: VA''s mail-service operation

A western blot assay was performed for the detection of Fasciola hepatica specific antigens for the diagnostic of fasciolasis; 72 sera were tested, 28 coming from patients with the parasitic disease and 44 from persons either healthy or presenting other diseases; 11 different antigenic bands were detected using sera from patients with fasciolasis. The 57 and 29 kDa specific antigens are considered like major, their specificity is about 100% and their respective sensibility, 79 and 93%. Band of 9-12 kDa is also appeared specific but is revealed only in 47% of the cases; 27 out of the 28 sera from patients with fasciolasis were able to recognize at least one of the 57, 29 or 9-12 kDa specific antigens. The present results suggest that western blot could be useful for the diagnosis of this parasitic disease as far as the criteria of positivity is based on the recognition of at least one of the major specific antigens.     

Isoforms of human recombinant follicle-stimulating hormone: comparison of effects on murine follicle development in vitro

The effects of three isoforms derived from recombinant human FSH on ovarian follicle development in vitro were characterized for the first time. The three subfractions comprised discrete pI ranges of 3. 6-4.6 (acid), 4.5-5.0 (mid), and 5.0-5.6 (least acidic). Follicular growth, estradiol secretion, and antral formation were assessed for each fraction of isoforms in a range of concentrations over a 5-day culture period. Least acidic FSH produced, at and above 1.5 ng/ml, a high percentage of follicles growing above the size threshold necessary for antral formation, whereas mid and acid FSH induced similar growth only at higher concentrations (7.5 ng/ml and 50 ng/ml, respectively). Least acidic FSH specifically induced the most rapid growth of follicles during preantral development. Acid FSH at all concentrations stimulated estradiol-17ss secretion later during culture and antral formation in a lower proportion of follicles than did least acidic and mid FSH. It can be concluded 1) that the least acidic isoform induced fastest preantral growth, producing the largest antral follicles at the lowest dose of all three fractions and 2) that the less and mid acidic isoforms had more impact on stimulation of estradiol production and antral formation than the acid isoform.     

Measurement of tritiated thymidine labeling index by incubation in vitro of surgically removed cervical cancer

Autoradiographic study was made on 24 cases of uterine cervical tumor, including 22 cases of squamous cell carcinoma, using a technique of in vitro labeling of surgical specimens with tritiated thymidine (3H-TdR) under hyperbaric oxygen. Labeling index of the cervical cancer ranged from 8.7 to 30.4% and was well correlated with the histological subtypes (spinal, transitional, and basal cell types). The upper limit of the growth fraction was also estimated from the general relationship between phase lengths. The necessity of sufficient oxygen tension for the S-phase cells to effect synthesis of DNA was stressed.     

Transglutaminase covalently incorporates amines into human immunodeficiency virus envelope glycoprotein gp120 in vitro

This is a retrospective review of 29 patients (33 hands) who underwent a palmaris longus transfer because of severe thenar atrophy secondary to median nerve entrapment at the wrist. The mean follow-up was 17 months. Ninety-four percent of our patients were satisfied because their thumb function improved. Twenty-six of the patients had the transfer at the time of initial release of the carpal tunnel, and three patients had the transfer when the carpal tunnel was released a second time. The transfer helps with thumb palmar abduction, and the palmaris longus is an expendable muscle for transfer.     

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.     

Retroviral pseudo-virus carrying the envelope proteins of neurotropic Friend murine leukemia virus effectively transferred retroviral vector into glial cells

We isolated the neurotropic Friend murine leukemia virus, FrC6 and its molecular clone A8, which proliferated in rat glial cell lines in vitro and in the rat brain in vivo. To investigate the contribution of viral envelope proteins to the neurotropism of A8 virus, the retroviral pseudo-virus carrying the envelope proteins of A8 virus and Moloney murine leukemia virus (MoMLV) was produced by transfecting the env gene of A8 virus (A8env) in the MoMLV based packaging cell, psi CRE. The phenotypically mixed pseudo-virus infected the rat glial cell lines as well as NIH 3T3 cells, whereas the psi CRE-produced pure pseudo-virus without A8env expression infected the glial cells at lower efficiency. Furthermore, the psi CRE cells with A8env expression produced pseudo-virus at a higher titer than normal psi CRE cells. The infectivity of the phenotypically mixed pseudo-virus to the glial cells was abolished by a neutralizing antibody against A8 virus, which did not reduce the ability of the psi CRE-produced pure pseudo-virus to infect NIH 3T3 cells. These results indicated that the envelope protein of A8 virus is assembled into the pseudo-viral particles and that it contributes to glial cell infection by the A8 virus.     

Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro

The effects of antioxidant-rich foods as adjuncts to a prudent diet were compared for 12 weeks in a randomized, single-blind and controlled trial in 204 (group A) and 202 (group B) patients with acute myocardial infarction. There was a significant decrease in cardiac end points in group A compared to group B (37 vs 58, p < 0.01) after 12 weeks. Within intervention group A, those 108 patients with greater adherence to the intervention program showed a greater reduction in cardiac end points (14 vs 58, p < 0.001), and a significant decrease in total mortality (6 vs 28, p < 0.001), including cardiac mortality (6 vs 25, p < 0.01) compared to group B. Underlying these beneficial effects, antioxidant-rich foods caused a significantly smaller rise in lactate dehydrogenase (LDH) cardiac enzyme in group A than in group B (427.8 vs 561.6 IU/dL), indicating that the protective influence of such a diet may be observed within 1 week. The subset of group A patients showing reduction in mortality also had a lesser rise in LDH and greater reduction in blood lipids, blood glucose and blood pressures. Antioxidant-rich foods also caused a significant decrease in blood lipids with a lower decrease in high-density lipoprotein cholesterol in group A than in group B. Assay of serum level of antioxidants and long-term follow-up may confirm our observations.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent

The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MG delta EGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MG delta EGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4.     

In vitro exposure of murine macrophages to ultraviolet light induces apoptosis

Ultraviolet light is a known exogenous stimuli with the ability to activate cell death by apoptosis. This study was done to examine the biologic effects of different energy levels of short wavelength UV light on cultured mouse macrophages. Cell proliferation, DNA content, and cellular ultrastructural architecture analysis demonstrated that ultraviolet light induces apoptosis in murine macrophages in culture. Exposure to 0.12J/cm2 evokes progressive cell demise with the classical features associated with apoptosis, whereas exposure to 5.0 J/cm2 results in extensive DNA degradation and crosslinking of cellular proteins. These two phenotypes are qualitatively described.