Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracil, 5-Azacytidine, and Ethyl Methane Sulfonate

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.     

Sialosyllactotetraosylceramide, a novel ganglioside antigen detected in human carcinomas by a monoclonal antibody

A novel ganglioside was detected in a small cell lung carcinoma by TLC-immunostaining of gangliosides with a monoclonal antibody, the C-50 MAb. Structural characterization showed this ganglioside to be IV3NeuAc-LcOse4Cer, a hitherto unknown ganglioside. This ganglioside has also been detected as a minor component in many different carcinomas using the C-50 MAb. The normally dominant CA-50 ganglioside antigen is IV3NeuAc, III4Fuc-LcOse4Cer. Based upon solid-phase binding to IV3NeuAc, III4-LcOse4Cer and IV3NeuAc-LcOse4Cer it is concluded that the C-50 MAb recognizes an epitope present in sialylated type I carbohydrate chains.     

Light-induced charge separation in Photosystem I at low temperature is not influenced by vitamin K-1

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

Production and Characterization of Monoclonal Antibodies to Barley Yellow Mosaic Virus and Their Use in Detection of Four Bymoviruses

Six monoclonal antibodies (MAbs) against a French isolate of barley yellow mosaic virus (BaYMV) pathotype 2 were produced and their isotypes determined. These MAbs were compared in ELISA for their reactivity with different isolates of BaYMV, wheat yellow mosaic virus (WYMV), wheat spindle streak mosaic virus (WSSMV) and oat mosaic virus (OMV).The six MAbs detected BaYMV in TAS ELISA and western blot, whereas in ACP ELISA no reaction was observed with isolates of BaYMV and WYMV. These MAbs could recognize the sequential motifs situated at the surface of viral particles. The six MAbs detected all the European isolates of BaYMV pathotype 1 and 2 and the Japanese isolate of this viral pathotype 1–1. In contrast to other MAbs, MAb IV did not react with the Japanese isolate of BaYMV pathotype II-l. In TAS ELISA. MAbs I, II, III, and IV detected the Japanese isolate of WYMV and American isolates of WSSMV only when they were captured by anti-WYMV polyclonal antibodies, A French isolate of OMV was detected only by the MAbs I and II in TAS ELISA with Polyclonal anti-BaYMV.     

Production and characterization of monoclonal antibodies against bovine pancreatic trypsin inhibitor

Two monoclonal antibodies (MAbs) have been produced, without the use of a supporting carrier, against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a mini-protein composed of 58 amino acids. Both MAbs obtained were found to be IgM. One of them was purified and further characterized. This MAb (ICI) binds to the immunogen with an association constant of 1.6 X 10(6)M-1 at pH 7.4. Competition experiments with trypsin or inactivated trypsin demonstrate that ICI MAb interacts with BPTI at, or near, the proteinase-binding site. ICI MAb binds, with a much lower association constant (approximately 200M-1), to an isoinhibitor (spleen inhibitor II) which differs from BPTI in seven amino-acids; three of these substitutions are at the active site, in the contact area with the proteinase.     

Analysis of cell-free human alpha1 integrin with a monoclonal antibody to the I-domain: detection in ocular fluid and function as an adhesion substrate

The alpha1 beta1 integrin, an inserted (1) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human alpha1beta1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human alpha1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV or alpha1 I-domain. MAb I B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free alpha1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.     

Photosynthetic reaction center of green sulfur bacteria studied by EPR

Membrane preparations of two species of the green sulfur bacteria Chlorobium have been studied by EPR. Three signals were detected which were attributed to iron-sulfur centers acting as electron acceptors in the photosynthetic reaction center. (1) A signal from a center designated FB, (gz = 2.07, gy = 1.91, gx = 1.86) was photoinduced at 4 K. (2) A similar signal, FA (gz = 2.05, gy = 1.94, gx = 1.88), was photoinduced in addition to the FB signal upon a short period of illumination at 200 K. (3) Further illumination at 200 K resulted in the appearance of a broad feature at g = 1.78. This is attributed to the gx component of an iron-sulfur center designated FX. The designations of these signals as FB, FA, and FX are based on their spectroscopic similarities to signals in photosystem I (PS I). The orientation dependence of these EPR signals in ordered Chlorobium membrane multilayers is remarkably similar to that of their PS I homologues. A magnetic interaction between the reduced forms of FB and FA occurs, which is also very similar to that seen in PS I. However, in contrast to the situation in PS I, FA and FB cannot be chemically reduced by sodium dithionite at pH 11. This indicates redox potentials for FA and FB which are lower by at least 150 mV than their PS I counterparts. The triplet state of P840, the primary electron donor, could be photoinduced at 4 K in samples which had been preincubated with sodium dithionite and methyl viologen and then preilluminated at 200 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.     

Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells.

Glycoprotein D (gD) is a viron envelope component of herpes simplex virus types 1 and 2. We have previously defined seven monoclonal antibody (MAb) groups which recognize distinct epitopes on the mature gD-1 protein of 369 amino acids. MAb groups VII, II, and V recognize continuous epitopes at residues 11-19, 272-279, and 340-356, respectively. MAb groups I, III, IV, and VI recognize discontinuous epitopes. Recent studies have focused on epitopes I, III, and VI. Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. A portion of discontinuous epitope I was located in a region within residues 233-275. For this study, we used recombinant DNA methods to create mutations in the gD-1 gene and studied the effects of those mutations on gD as expressed in mammalian cells. Plasmid pRE4, containing the coding sequence of gD-1 and the Rous sarcoma virus long terminal repeat promoter, was transfected into mammalian cells. The expressed protein, gD-1-(pRE4), was identical in size and antigenic properties to gD-1 from infected cells. Six in-frame deletion mutations were subsequently constructed by using restriction enzymes to excise portions of the gD-1 gene. Plasmids carrying these mutated forms were transfected into cells, and the corresponding proteins were examined at 48 h posttransfection for antigenicity and glycosylation patterns. Three deletions of varying size were located downstream of residue 233. Analysis of these mutants showed that amino acids within the region 234-244 were critical for binding of DL11 (group I), but not for other MAb groups. Three other deletion mutants lost all ability to bind MAbs which recognize discontinuous epitopes. In addition, much of the gD expressed by these mutants was observed to migrate as high-molecular-weight aggregated forms in nondenaturing gels. Each of these mutations involved the loss of a cysteine residue, suggesting that disulfide linkages play an essential role in the formation of discontinuous epitopes. The extent of glycosylation of the mutant gD molecules accumulated at 48 h posttransfection suggested altered carbohydrate processing. In one case, there was evidence for increased O-linked glycosylation. Those proteins which had lost a cysteine residue as part of the deletion did not accumulate molecules processed beyond the high-mannose stage. The results suggest that carbohydrate processing during synthesis of gD is very sensitive to alterations in structure, particularly changes involving cysteine residues.     

Monoclonal antibodies against bovine type IX collagen (LMW fragment): production, characterization, and use for immunohistochemical localization studies.

Four high-affinity monoclonal antibodies (MAb) which react specifically with the low molecular weight (LMW) fragment of bovine type IX collagen (BIX) have been produced in mice. On the basis of the ability of these MAb to cross-react with type IX collagen purified from human, rat, and chick cartilage and to inhibit one another in a competitive inhibition assay, we conclude that the MAb D1-9, B3-1, and B2-7 recognize unique epitopes, whereas MAb B4-5 recognizes the same epitope as B3-1. None of the MAb reacted with bovine type I, II, and XI collagen. MAb D1-9 and B3-1 were tested for their ability to bind to tissue antigen, using an immunohistochemical assay system. Positive immunoperoxidase reactions were observed in the perichondrocytic regions of human and rat costochondral cartilage. Positive responses were also detected in rat auricular cartilage, as well as in tissue obtained from the middle and inner ears of rats and mice. This report demonstrates the relative ease of producing MAb to heterologous type IX collagen and the utility of these MAb for localizing type IX collagen in cartilage and cartilage-like tissues.     

Characterization of apolipoprotein B-containing lipoproteins separated by preparative free flow isotachophoresis

Preparative free flow isotachophoresis (ITP) was used for the fractionation of apoB-containing lipoproteins (d less than 1.063 g/ml) from fasting and postprandial sera derived from normolipidemic individuals. According to their net electric mobility, four major particle groups (I-IV) have been recognized. The fast-migrating particles in group I, which correspond predominantly to very low density lipoproteins (VLDL), are rich in triglycerides, free cholesterol, phosphatidylcholine, and apoE and C apolipoproteins. This group expresses nonspecific binding to fibroblasts but binds to HepG2 cells with high affinity (KD = 3.6 micrograms/ml, Bmax = 37 ng) to a single class of binding sites. The particles migrating in group II, which are related to intermediate density lipoproteins (IDL), are richer in cholesteryl esters and apoB than those in group I. They interact specifically with a single site on fibroblasts (KD = 7.8 micrograms/ml, Bmax = 54 ng) while on HepG2 cells two binding sites, one with a higher (KD = 3.5 micrograms/ml, Bmax = 22 ng) and one with a lower affinity component (KD = 16.9 micrograms/ml, Bmax = 53 ng), are involved. The particles migrating in groups III and IV correspond to low density lipoproteins (LDL). The protein moiety of both fractions consists almost exclusively of apoB. Group III represents cholesteryl ester-rich LDL particles, while the particles in group IV contain smaller amounts of cholesteryl esters. The lipoproteins of both groups are ligands for apoB,E-receptors. However, the particles in group IV interact with fibroblasts with the highest affinity (KD = 2.3 micrograms/ml, Bmax = 58 ng) and with the biphasic HepG2 cell binding sites with the lowest affinity of all analyzed groups (KD1 = 11.2 micrograms/ml, Bmax1 = 58 ng, KD2 = 68 micrograms/ml, Bmax2 = 170 ng). When apoB-containing lipoproteins were isolated from postprandial sera of the same individuals, significant changes in the lipid composition were observed only in particle groups I and II, where the triglyceride and phospholipid content was enhanced. Group I particles from postprandial serum bind to HepG2 cells with a higher affinity (KD = 2.5 micrograms/ml) than group I particles from fasting serum. Postprandial group II particles bind with the same affinity to the biphasic HepG2 cell receptor as fasting group II particles, while the affinities of postprandial group III (KD1 = 4.1 micrograms/ml, KD1 = 47 micrograms/ml) and group IV particles (KD1 = 3.9 micrograms/ml, KD2 = 38 micrograms/ml) to the high affinity binding site of the biphasic receptor are enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.     

Characterization of urinary volatiles in Swiss male mice (<Emphasis Type="Italic">Mus musculus</Emphasis>): bioassay of identified compounds

The present study was carried out to investigate the chemical nature of the urine of male mice and to assess its bioactivity. Urine of mature male mice was extracted with dichloromethane (1:1 ratio v/v) and analysed by gas-chromatography linked mass-spectrometry (GC-MS). Ten different compounds such as alkanes, alcohols, etc. were detected in the urine. Among the ten, five compounds are specific to males, namely 3-cyclohexene-1-methanol (I), 3-amino-s-triazole (II), 4-ethyl phenol (III), 3-ethyl-2,7-dimethyl octane (IV) and 1-iodoundecane (V). The compound, 4-ethylphenol, has been previously reported in several strains of male mice. Furthermore, the compounds (II) and (IV) are similar to 2-sec-butylthiazole and dehydro-exo-brevicomin compounds which have already been reported in male mice. Bioassay revealed that compounds (II), (III) and (IV) were responsible for attracting females and in inducing aggression towards males, as compared to the other compounds, i.e. (I) and (V). The results indicate that these three volatiles (II, III and IV) of male mice appear to act as attractants of the opposite sex.     

Identification of functional regions of herpes simplex virus glycoprotein gD by using linker-insertion mutagenesis.

Glycoprotein gD is a component of the herpes simplex virus (HSV) envelope essential for virus entry into susceptible cells. Previous studies using deletion and point mutations identified a functional domain of HSV-1 gD (gD-1) from residues 231 to 244. However, many of the deletion mutations had global effects on gD-1 structure, thus precluding assessment of the functional role of large portions of the protein. In this study, we constructed a large panel of linker-insertion mutants in the genes for gD-1 and HSV-2 gD (gD-2). The object was to create mutations which would have only localized effects on protein structure but might have profound effects on gD function. The mutant proteins were expressed in transiently transfected L cells. Monoclonal antibodies (MAbs) were used as probes of gD structure. We also examined protein aggregation and appearance of the mutant glycoproteins on the transfected cell surface. A complementation assay measured the ability of the mutant proteins to rescue the infectivity of the gD-null virus, FgD beta, in trans. Most of the mutants were recognized by one or more MAbs to discontinuous epitopes, were transported to the transfected cell surface, and rescued FgD beta virus infectivity. However, some mutants which retained structure were unable to complement FgD beta. These mutants were clustered in four regions of gD. Region III (amino acids 222 to 246) overlaps the region previously defined by gD-1 deletion mutants. The others, from 27 through 43 (region I), from 125 through 161 (region II), and from 277 to 310 (region IV), are newly described. Region IV, immediately upstream of the transmembrane anchor sequence, was previously postulated to be part of a putative stalk structure. However, residues 277 to 300 are directly involved in gD function. The linker-insertion mutants were useful for mapping MAb AP7, a previously ungrouped neutralizing MAb, and provided further information concerning other discontinuous epitopes. The mapping data suggest that regions I through IV are physically near each other in the folded structure of gD and may form a single functional domain.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Linkage of reduced receptor affinity and superinfection to pathogenesis of TR1.3 murine leukemia virus

TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in the K(D) of TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology.     

Fumonisin B1 inhibits mitochondrial respiration and deregulates calcium homeostasis--implication to mechanism of cell toxicity

Fumonisin B(1) (FB(1)) is a neurodegenerative mycotoxin produced by Fusarium verticiloides mould that contaminates maize worldwide. FB(1) toxicity has been connected with deregulation of sphingolipid metabolism, but the mechanism of cytotoxicity remains controversial. In cell cultures of rat primary astrocytes and human neuroblastoma (SH-SY5Y), we found that FB(1) inhibits mitochondrial complex I, which leads to a decrease in the rate of mitochondrial and cellular respiration, depolarisation of the mitochondrial membrane, induction of reactive oxygen species (ROS) production in mitochondria and deregulation of calcium signalling. Despite the increase in ROS production, the intracellular level of glutathione (GSH) was significantly increased. After 24h of FB(1) exposure, no cell death was observed. Thus, mitochondria appear to be the primary target of FB(1), which leads to sustained deregulation of calcium homeostasis and presumably to cell death.