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In an amyotrophic lateral sclerosis (ALS) patient who also had an IgA gammopathy, autopsy studies identified the IgA in the surviving motor neurons. Further, the IgA bound the surface of isolated bovine motor neurons and inhibited neuronal proliferation in culture. To determine the pathologic basis of this IgA interaction with motor neurons, a neuroblastoma cDNA library was generated and screened with the IgA monoclonal antibody. Reactive clones were identified as flavin adenine dinucleotide (FAD) synthetase. To extend this finding to ALS in general, quantitative RT-PCRs were performed on blood samples from 26 ALS and 30 control blood samples to determine mRNA expression levels of FAD synthetase and other electron transport chain proteins, specifically riboflavin kinase (RFK), cytochrome C1 (CYC1), and succinate dehydrogenase complex subunit B (SDHB). All expression levels were measured against a control enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression levels for a non-respiratory chain protein (beta-actin) were also measured. We found that FAD synthetase expression levels were decreased in ALS samples compared to expression levels in controls (P = 0.0151). Expression levels for RFK, CYC1, and SDHB were also significantly decreased in the ALS group (P = 0.0025, P = 0.0002, and P < 0.0001, respectively). As control, expression levels for beta-actin did not show a significant difference between ALS and control groups (P = 0.2118). Our data show that a reduction in electron transport proteins, namely FAD synthetase, RFK, CYC1, and SDHB, is seen in patients with ALS. It is possible that this may have an effect on oxygen-dependent metabolic pathways. Human motor neurons may be particularly susceptible to injury if there is sub-optimal oxidative metabolism.

Polyclonal antibody/carbon nanotube (CNT) conjugates have been prepared through a di-imide activated amidation process using partially oxidized CNTs. The antibody/nanotube conjugate was used as a microwave absorber with selective affinity for the cell receptor CD44, overexpressed on the surface of PC3 prostate cancer cells. Unlike the most common protocols involving thermic ablation by infrared radiation, microwaves offer a significant higher penetration depth, with instant localized heating when combined with CNTs. The heating rates obtained when multi-walled CNT suspensions in buffer are exposed to microwaves is reported. The antibody/nanotube conjugates were tested in a series of in vitro experiments in which the microwave power and the exposure time were consistently varied. After MTS assays, optimized conditions have been established for the thermal ablation of PC3 cells, while healthy cells remained unharmed. The trends observed in vitro were validated in vivo, using zebrafish embryos as an animal model.

Taxol, rii y extrced fro the bark of the western yew, Taxus brenfolia, is reportedly the first of a new class of anti-cancer agent. It Taxol, reportedly the first of a new class of anti-cancer agents, was originally extracted from the bark of the western yew, Taxus brevifolia (1). In vivo, it prevents the disassembly of microtubules, causing the arrest of cells at the G2/M step of the cell cycle (2). Its mechanism differs from that of other anti-cancer agents that have tubulin as their target, such as colchicine, podophyllotoxin, or vinblastine, which inhibit tubulin polymerization (3). The site of action of taxol on tubulin is being investigated in several laboratories. Photoaffinity-labeling experiments implicate the N-terminal region of (3-tubulin as the site of attachment (4, 5).With the aim of using immunological techniques to investigate the action of taxol, we sought to prepare an anti-idiotypic antibody that mimics taxol, using an auto-anti-idiotypic strategy (6, 7). We have succeeded in obtaining an antibody, 82H, that, as intact antibody or Fab fiagment, mimics taxol in its tubulinmicrotubule interactions to the extent that it can cause tubulin to assemble into microtubules. MATERIALS AND METHODSPreparation of and Screening for Anti-Idiotypic Antibodies. The object was to screen for monoclonal antibodies that showed taxol-inhibitable binding to Fab fragments ofaffinitypurified rabbit anti-taxol antibody R585 (8).Preparation of Fab fragments of R585. Rabbit anti-taxol antiserum R585 (8) was affinity-purified as follows. Two affinity columns were prepared. Either 10 mg of bovine serum albumin (BSA) or 10 mg of taxol covalently linked to rabbit serum albumin (taxol-RSA) (8) was dissolved in 1 ml of 0.1 M 2-(N-morpholino)-ethanesulfonic acid (Mes) buffer (pH 4.8) and added to 2 ml of Affi-Gel 10 (Bio-Rad). The mixtures were rocked gently overnight at 40C. The gels were then washed with several column volumes of phosphatebuffered saline (PBS, pH 7.3) and with 0.2 M glycine hydrochloride buffer (pH 2.5) and then equilibrated with PBS. Six milliliters of R585 antiserum was first recirculated through the taxol-RSA-Affi-Gel 10 column overnight at 40C at a rate of0.2 ml/min. The column was then extensively washed with PBS at a rate of 1.0 ml/min to remove all nonspecifically bound proteins. The antibodies were then eluted with 0.2 M glycine hydrochloride (pH 2.5) at a rate of 0.5 ml/min. The eluted antibodies were immediately neutralized with 2 M Tris base and dialyzed against PBS at 40C overnight. On the following day, the eluted antibodies were recycled through the BSA-Affi-Gel 10 column at 40C overnight to remove BSA-binding antibodies. The effluent, which contained antitaxol antibodies in PBS, was collected and concentrated by a Centriprep-10 concentrator (Amicon) to a volume of 1-2 ml.Fab fragments were prepared from the affinity-purified antibodies as described (9).Screening for anti-idiotypic antibodies. A competitive ELISA was used to assay for taxol-inhibitable binding to the Fab fiagments of ...

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In the current study, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR were used to clone full-length putative Na(+)-H(+) exchanger isoforms (NHE2a) cDNA from the gills of Fundulus heteroclitus. The 2480 bp cDNA includes a coding region for a protein that shows a 57% amino acid homology to rabbit NHE2. These sequences allowed data mining of available fish genome data, which revealed at least three NHE2 subtypes in some teleost species.

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Human immunodeficiency virus type 1 is unique among retroviruses in that infectivity requires specific incorporation into virions of the cellular protein cyclophilin A through interactions with the Gag polyprotein. Here we show that monoclonal antibody B11 1.4, which recognizes a cyclophilin-binding epitope on cyclosporine, detects denatured or native human immunodeficiency virus type 1 capsid. B11 1.4 does not recognize the capsids of other retroviruses, and binding is inhibited by cyclosporine or by cyclophilin A.