We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.

Acetylthiocholine↗

PubMedDate not supplied

Localization of a novel adhesion-promoting site on acetylcholinesterase using catalytic antiacetylcholinesterase antibodies displaying cholinesterase-like activity.

A monoclonal antibody (MAb) raised against human acetylcholinesterase was found to have catalytic activity. A similar phenomenon was observed in a polyclonal antibody raised against the same antigen. The antibodies were demonstrated to be pure, and no contamination with either acetylcholinesterase or butyrylcholinesterase was found. Both antibodies hydrolyzed acetylthiocholine, an acetylcholinesterase substrate, and the MAb followed Michaelis-Menten kinetics. Six other MAbs and one other polyclonal antibody showed no evidence of catalytic activity. Acetylcholinesterase is a key component in the transmission of the nerve impulse, and is also expressed nonsynaptically during embryonic development, and abnormalities in expression are seen in neural tumors and degenerative disorders. This unusual expression is believed to be associated with a novel function of the enzyme related to differentiation and cell adhesion. Autoantibodies to acetylcholinesterase have been observed in a variety of neurologic, muscular, and autoimmune disorders. In an investigation of the possible role of acetylcholinesterase in cell adhesion, we showed that the enzyme promoted neurite outgrowth in neuroblastoma cell lines, and conversely, that certain antiacetylcolinesterase antibodies abrogated cell-substrate adhesion. Interestingly, the antibodies most effective in this regard were catalytic. Preliminary epitope analysis indicated a conformational epitope in the N-terminal domain. This domain contains the active site within a deep gorge and the peripheral anionic site at the rim of the gorge. Peripheral-site inhibitors, but not active-site inhibitors, also interfered with adhesion, and competed with the catalytic monoclonal binding to acetylcholinesterase, indicating that the epitope recognized is associated with the peripheral anionic site. The inhibitor data also support the supposition that catalysis in these antibodies may have arisen from stable complexation of acetylcholinesterase with an inhibitor. We conclude that the catalytic antiacetylcholinesterase antibody interacts with structures associated with the peripheral anionic site, thus defining a novel site on the molecule involved in cell adhesion. This finding has implications for our understanding of the potential importance of this peripheral site in a variety of congenital, neoplastic, and degenerative conditions.

Acetylcholinesterase↗

PubMedDate not supplied

Splénic artery aneurysm.

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Adolescent↗

PubMedDate not supplied

Long-term effects of strokes on bone mass.

The purpose of the study was to determine the long-term effects of muscle weakness secondary to strokes on the bone mineral content of the hemiparetic limb. Patients who had experienced single recent strokes were studied. The bone mineral content of each limb was measured by Dual Energy X-ray Absorptiometry using the region of interest analysis program. Muscle strength of each muscle group was ranked using the Oxford scale, and the mean was calculated for each limb. Bone and muscle parameters were measured within seven days after the stroke and repeated thereafter at monthly intervals for up to 6 mo. A repeated measures analysis of variance, Newman-Keuls pair-wise comparisons, and orthogonal contrasts were done for each parameter. Significance levels were set at P < 0.05. Sixteen patients were included in this study. Demineralization was more pronounced in the upper than lower limbs. Demineralization of bones on the paralyzed side started during the first month after the stroke and gradually progressed. By the fourth month, the bone mineral content decreased by a mean of 9.3% (P = 0.01) and 3.7% (P = 0.01) in the upper and lower limbs, respectively, for the 11 patients followed for 4 mo. In the patients we followed for more than 4 mo, there was no further significant mineral loss. No change in bone mineral content was observed in the healthy nonparetic limbs. In conclusion, after a stroke, bone demineralization occurs in the paralyzed side and reaches its maximum within 3 to 4 mo. Arms are affected more than legs.

Absorptiometry, Photon↗

PubMedDate not supplied

Subarachnoid microcatheter anesthesia in small children.

BACKGROUND AND OBJECTIVES: Standard single dose spinal anesthesia is a recognized technique for neonates and small children. The advent of the subarachnoid microcatheter technique potentially expands the use of spinal anesthesia. The authors studied the use of repeated neuroblock via spinal catheters in 10 children under 5 years, undergoing major abdominal surgery of 2 hours or longer. METHODS: The children had a mean weight of 8 +/- 1.8 kg and a mean age of 12.5 +/- 5.7 months. General anesthesia was administered as isoflurane 1%-1.5% in a 50% oxygen in air mixture via an endotracheal tube connected to an Ohmeda ventilator. A Kendall Cospan microcatheter pack was used to place a 28-gauge catheter via a 22-gauge needle at the L4-5 or L5-S1 interspace. Isobaric plain bupivacaine 0.5% was injected at 0.2 mL/kg, up to a maximum of 1 mL. This was repeated whenever the pulse or systolic blood pressure rose above 20% of baseline values. Neurologic examinations for motor or sensory deficits were done 24 hours after the block, at discharge, and 1 month later. RESULTS: The catheters were easily placed and removed without difficulty. Surgical conditions were excellent and, as judged by pulse and blood pressure, analgesia during surgery was optimum. The mean duration of the neuroblock was 59.7 +/- 2.1 minutes. Systolic blood pressure decreased initially by a mean of 8% (6.5 +/- 0.7 mmHg) and after follow-up doses by 2%-6% below baseline values. On recovery, analgesic levels were T1-T6. Three children had the catheters left in situ for postoperative analgesia. All three leaked 2-3 mL of clear glucose-containing fluid alongside the catheter overnight. No neurologic deficits were seen. CONCLUSIONS: The technique was a very effective method of providing adjunct operative anesthesia in infants and small children. Analgesia, muscle relaxation, and cardiovascular stability were present and no complications arose besides the apparent cerebrospinal fluid leak. The resultant risk of infection or possible duracutaneous fistula formation contraindicates the use of subarachnoid catheters of 28 gauge for postoperative analgesic use in young children.

Abdomen↗