A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility to Influenza Virus In Vitro

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10–100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.     

Phenolic Compounds in Stems of Sunflower Plants Inoculated with Sclerotinia sclerotiorum and their Inhibitory Effects on the Fungus

Sunflower plants at I (vegetative), II (closed flower-buds) and III (open flower-buds) growth stages were experimentally inoculated with Sclerotinia sclerotiorum. At the 5th day after inoculation, all plants from I, III and 70 % of II (II_T) did not present wilt symptoms while 30 % of age II plants (II_s) presented severe wilt symptoms in all their leaves and had collapsed. Total soluble phenols were measured in the “lesion” (L) and its “healthy” (H) surrounding zones of all inoculated and control plants. A significant increase in phenols was found in the L and H zones of tolerant plants (I, II_t and III). The corresponding methanolic extracts showed a strong inhibitory effect on the mycelial growth (MG) of the fungus. On the other hand no statistically significant increase took place in the L zones from susceptible plants; their methanolic extracts did not inhibit MG. The chemical analysis of methanolic extracts showed that the isochlorogenic acid was the more inhibitory compound. It significantly accumulated in the L of tolerant plants but did not in those of susceptible ones.     

The In Vitro Mass-Produced Model Mycorrhizal Fungus,Rhizophagus irregularis,Significantly Increases Yields of the Globally Important Food Security Crop Cassava

The arbuscular mycorrhizal symbiosis is formed between arbuscular mycorrhizal fungi (AMF) and plant roots. The fungi provide the plant with inorganic phosphate (P). The symbiosis can result in increased plant growth. Although most global food crops naturally form this symbiosis, very few studies have shown that their practical application can lead to large-scale increases in food production. Application of AMF to crops in the tropics is potentially effective for improving yields. However, a main problem of using AMF on a large-scale is producing cheap inoculum in a clean sterile carrier and sufficiently concentrated to cheaply transport. Recently, mass-produced in vitro inoculum of the model mycorrhizal fungus Rhizophagus irregularis became available, potentially making its use viable in tropical agriculture. One of the most globally important food plants in the tropics is cassava. We evaluated the effect of in vitro mass-produced R. irregularis inoculum on the yield of cassava crops at two locations in Colombia. A significant effect of R. irregularis inoculation on yield occurred at both sites. At one site, yield increases were observed irrespective of P fertilization. At the other site, inoculation with AMF and 50% of the normally applied P gave the highest yield. Despite that AMF inoculation resulted in greater food production, economic analyses revealed that AMF inoculation did not give greater return on investment than with conventional cultivation. However, the amount of AMF inoculum used was double the recommended dose and was calculated with European, not Colombian, inoculum prices. R. irregularis can also be manipulated genetically in vitro, leading to improved plant growth. We conclude that application of in vitro R. irregularis is currently a way of increasing cassava yields, that there is a strong potential for it to be economically profitable and that there is enormous potential to improve this efficiency further in the future.     

Calcium-Activated-Calcineurin Reduces the In Vitro and In Vivo Sensitivity of Fluconazole to Candida albicans via Rta2p

Due to the emergence of drug-resistance, first-line therapy with fluconazole (FLC) increasingly resulted in clinical failure for the treatment of candidemia. Our previous studies found that in vitro RTA2 was involved in the calcineurin-mediated resistance to FLC in C. albicans. In this study, we found that calcium-activated-calcineurin significantly reduced the in vitro sensitivity of C. albicans to FLC by blocking the impairment of FLC to the plasma membrane via Rta2p. Furthermore, we found that RTA2 itself was not involved in C. albicans virulence, but the disruption of RTA2 dramatically increased the therapeutic efficacy of FLC in a murine model of systemic candidiasis. Conversely, both re-introduction of one RTA2 allele and ectopic expression of RTA2 significantly reduced FLC efficacy in a mammalian host. Finally, we found that calcium-activated-calcineurin, through its target Rta2p, dramatically reduced the efficacy of FLC against candidemia. Given the critical roles of Rta2p in controlling the efficacy of FLC, Rta2p can be a potential drug target for antifungal therapies.     

Characterization of the anti-fungal activity of a Bacillus spp. associated with sclerotia from Sclerotinia sclerotiorum

Sclerotinia sclerotiorum fruiting bodies (sclerotia) were found to harbour bacteria that possess anti-fungal activity. Among 1,140 bacterial isolates collected, 32 were found to inhibit the growth of four common fungal pathogens of canola, S. sclerotiorum, Rhizoctonia solani, Alternaria brassicae and Leptosphaeria maculans. One of these broad-spectrum isolates, LEV-006, was found to be closely related to Bacillus subtilis based on 16S rRNA analysis. The anti-fungal activities were purified and found to be associated with a low molecular weight peptide complex consisting mostly of the cyclic lipopeptide fengycin A and B, as revealed by matrix-assisted laser desorption/ionization time-of-flight and post-source decay analysis, as well as two proteins of 20 and 55 kDa. Peptide mass fingerprinting revealed that the 55-kDa protein was similar to vegetative catalase 1; however, when the enzyme was expressed in Escherichia coli, it exhibited catalase but not anti-fungal activity. The sequences of several peptides from the 20-kDa protein were obtained and indicated that it was a unique anti-fungal protein.     

应用二维液相色谱评价低毒病毒感染对板栗疫病菌总蛋白表达的影响

根据丝状真菌自身的特点,本文建立了一套完整的适合于板栗疫病菌蛋白质组分析的总蛋白质提取方法,并使用二维液相系统对野生强毒株EP155和受低毒病毒感染的弱毒株EP713进行了差异蛋白质组的比较。用ProteoVue和DeltaVue软件进行分析,得到了重复性较好的二维模拟凝胶图谱,在EP155和EP713之间共找到296个峰值差异强度在2倍以上的蛋白质紫外吸收峰:以EP155为标准,病毒感染导致蛋白上调的209个,下调的87个。根据蛋白质分子量,使用SDS-PAGE聚丙烯酰胺凝胶电泳可以得到纯度更高的蛋白质条带,本研究为大规模质谱鉴定丝状真菌差异表达蛋白质提供了一种新方法。     

Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Ss-Sl2, a novel cell wall protein with PAN modules, is essential for sclerotial development and cellular integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum.     

The Jak2 Small Molecule Inhibitor,G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo

Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Immunization of Chlamydia pneumoniae (Cpn)-Infected Apobtm2SgyLdlrtm1Her/J Mice with a Combined Peptide of Cpn Significantly Reduces Atherosclerotic Lesion Development

Objective

To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn.

Materials and Methods

Six-week-old Apob^tm2SgyLdlr^tm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants.

Results

Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells.

Conclusions

Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.     

Development of a Screening Assay for the In Vitro Evaluation of Thromboxane A2 Synthase Inhibitors

Abstract

Inhibitors of thromboxane A, (TxA₂) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA₂ synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA₂ synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA₂ synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA₂ synthase be performed.     

Comparison of responses of ascospores and mycelium by ELISA with anti-mycelium and anti-ascospore antisera for the development of a method to detect Sclerotinia sclerotiorum on petals of oilseed rape

The goal of this project was the development of a serological test for the detection of Sclerotinia sclerotiorum on oilseed rape petals. Since the fungus exists in two forms on petals, as ascospores and mycelium, the responses of anti-mycelium and anti-ascospore antisera against these two kinds of antigens were compared. Two anti-mycelium sera, Smy and Smy', were produced against mycelial soluble extracts at different concentrations (0.3 mg and 0.1 mg of protein ml^-1). Smy gave the greatest response level with eight S. sclerotiorum mycelial extracts tested. It had a very superior level of recognition to that of anti-ascospore serum, Ssp, when mycelium was tested as antigen. In contrast, Ssp and Smy were equally reactive when exposed to ascospores but the sensitivity of the assay was low. For each antiserum, the solution from which ascospores had been removed reacted similarly to the original suspension containing ascospores. A collection of fungi likely to be found on oilseed rape petals was examined. Cross-reactions were produced with both antisera, especially with Botrytis cinerea for which greater cross-reactivities were produced with Smy. The cross-absorption of antiserum Smy with a mycelial extract of B. cinerea considerably reduced this cross-reaction. The choice of antiserum for the development of a reliable detection system is discussed.     

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.     

Effect of a Single Dose of β,β''-Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro

The biochemical properties of neurofilaments isolated from control and iminodipropionitrile-treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross-linking between neurofilament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile-treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament-associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilament-associated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament-associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.