Neurofilaments (also called 10-nm filaments or intermediate filaments) from bovine brain were incubated with microtubule protein at 37"C in the presence or absence of 1 mM ATP and in a buffer that allowed microtubule assembly. Fallingball viscometry revealed that the (non-Newtonian) apparent viscosity of the ATP-containing mixtures is 5-20 times greater than that of the mixtures prepared without ATP. A larger ATP-dependent increase in viscosity (approximately 100-fold) was seen when purified tubulin replaced microtubule protein. The (1,4,5), and occasional observations of highly ordered arrays of neurofilaments surrounding microtubules (6) suggest together that the two structures are connected in vivo as part of an axoplasmic filamentous network. Neurofilaments are transported in the slow component of axonal transport (7,8), and it has been suggested (7) Neurofilaments, Tubulin, and Microtubule-Associated Proteins (MAPs). Neurofilaments were prepared from bovine brain as described (17). Microtubule protein referred to as "threetimes-cycled" was prepared by three cycles of the assembly/ disassembly method of Shelanski and coworkers (14, 18). Phosphocellulose (PC)-purified tubulin was prepared from twicecycled microtubule protein (19,20). The fraction that was initially retained by the PC column was eluted in buffer A containing 0.8 M NaCl and centrifuged for 60 min at 95,500 X g to remove any neurofilaments (see ref. 15). This material is referred to as "whole MAPs. " Aliquots of the whole MAP fraction were heated to 100°C for 5 min and then were centrifuged at 4°C for 20 min at 25,000 X g to remove flocculated protein (21). This material is referred to as "heat-treated MAPs." Purified MAP2 was prepared as described by Kim et al. (22). Upon NaDodSO4/polyacrylamide gel electrophoresis by the method of Laemmli (23), each of these five preparations appeared quite similar in protein composition to those described in the references given. The tubulin polymerized in buffer A at protein concentrations greater than about 2.7 mg/ml. Each preparation was dialyzed against buffer A and then stored in liquid N2. Aliquots were thawed, centrifuged for 10 min in a clinical centrifuge at 4°C, and used within 6 hr.Viscometry. Falling-ball viscometers were constructed and employed exactly as described by . Protein solutions were mixed at 4°C, and appropriate additions were made of nucleotide triphosphate solutions or of equal volumes of buffer in the cases of the controls. The cold solutions were immediately drawn into the viscometers, which were then immersed in a Tamson water bath controlled to +0.010C at 37. 0°C. The mixing and loading procedure required Abbreviations: MAP(s), microtubule-associated protein(s); PC, phosphocellulose; AdoPP[NH]P, adenyl-5'-yl imidodiphosphate. t Present address:

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Patients with recurrent spontaneous abortions have been successfully treated in many centers with third-party immunization directed to a putative TLX antigen system. This immunotherapy requires the screening of a large number of donors to match the patients' red blood cell (RBC) phenotype and has the potential risks associated with transfusions from 30 to 50 donors. Our modified approach to third-party immunization is to use irradiated frozen-stored purified lymphocytes pooled from five normal donors. Mononuclear cells from normal donors are obtained in a cell separator. After sedimentation and Ficoll-Hypaque separation, the cells are stored in liquid N2. The RBC depletion of the final preparation is of the order of 5 to 6 logs, theoretically decreasing the need for RBC phenotyping except for the Rh system. Using a highly sensitive fluorescence-activated cell sorter technique and an ADCC assay, we found that ABH, Rh, Fya Fyb, Jka Jkb, MNS, and Kell antigens are either not expressed by cryopreserved human mononuclear cells, or, if so, they are below the level of detection of these highly sensitive assays. We conclude that the use of pooled frozen mononuclear cells is an adequate alternative for immunotherapy. It decreases the transfusion risks associated with exposure to a large number of donors and the need for RBC phenotyping, making this modality of treatment more accessible.