Richard D. Feinman

The Proteinase-Binding Reaction of α2M

First identified as a proteolytic enzyme inhibitor, a2-macroglobulin (azM) has assumed new importance because of the nature of its cellular receptor and its potential role as a transport protein for growth factors and other agents. Most of the receptor interactions and transport functions of a2M, however, depend on prior reaction with proteinases, and an understanding of proteinase binding remains at the heart of azM physiology. The general features of the proteinase reaction are understood, but a complete description has so far been elusive-a general rate equation has not been obtained and, in some cases, the number and the nature of the intermediates are unknown. The mechanism of binding of proteinases to azM is unusual; the active site of the proteinase remains free and, in this way, the mechanism is very different from that of the classical proteinase inhibitors such as the serpins’” or the plant and pancreatic inhibitors! However, the small protein inhibitors and a2M have one feature in common: they contain a reactive site, referred to as the “bait” region in azM, with a susceptible peptide bond that provides a target for the proteinase. In the case of the serpins or plant inhibitors, the reaction at this site stops before proteolysis is complete. A stable complex is formed that, in most cases, is a distorted tetrahedral inte~mediate.~ For azM, on the other hand, bait region cleavage is successful and the active site of the bound proteinase remains free. Proteinase binding occurs through amide bonds between its lysyl amino groups and a glutamyl residue of the unique thioester bond of a2M (equation 1) and through conformational changes that sterically encapsulate (“trap”) the proteinase. Although free to react with small substrates and inhibitors, the active site of the bound proteinase is prevented by the steric constraints of the trap from reaction with large protein substrates or protein inhibitors:

Operant punishment of eye elevation in the green crab, Carcinus maenas

Extension of the eye after reflex withdrawal was suppressed by punishing each extension with a brief puff of air. Experimental animals showed a decrease in the rate of responding, and an increase in the latency to the next response during 30-min sessions. The effect of punishment per se was controlled for by the use of yoked animals that received punishments whenever the experimental (master) animals did. This control group did not show the increased latency, and kept the eye erect for most of the session. Experiments were performed with pairs of animals, one eye of each used as master or control, or, alternatively, with single animals in which one eye served as the yoked control for the other. This latter group gave more reliable and reproducible differences between master and yoke than the pairs of animals. Retention was tested by subjecting animals to three sessions separated by a 12-hr rest. The results indicated some savings but this was not a dramatic effect. To demonstrate that the learning was operant in nature, that is, that it depended on the contingency between the act of eye extension and punishment, experiments were performed in which a delay was introduced between the response and the onset of punishments. A delay of 20 s was found to completely eliminate the learned suppression: animals showed latencies close to that of naive animals and responded at a constant high rate throughout the session. Delays of 10, 5, and 2.5 s were found to have a decreasing effect on the learning, and a delay of 1.25 s produced behavior that was within experimental error of that of animals subjected to immediate punishment.

Lever-press conditioning in the crab

An operant chamber has been developed for studying lever-press conditioning in the green crab Carcinus maenas. In one series of experiments, animals were presented with a single bar and were reinforced with food for every bar press. Performance increased over time and high rates of responding were observed after 2 days of training. The response rate was always higher than that for a yoked (noncontingent) control group. When the contingencies were switched, the animals adjusted to the new conditions. Discrimination in the lever-press apparatus was demonstrated in a second experiment in which crabs had to choose between two bars, one (S+) caused food to be dispensed while the other (S-) was inactive. Experimental animals pressed the S+ bar at a significantly higher rate than the S- bar. When the contingencies associated with the lever were reversed, animals learned to switch to the correct bar by the second day. It was not necessary to reinforce every response: animals maintained high rates of responding on a schedule where every other response was reinforced. Animals used different methods of pressing the bar; the most common was extension of the claw, predominantly at the meropodite-carpopodite joint.

Insulin, carbohydrate restriction, metabolic syndrome and cancer

We propose that dietary carbohydrate restriction, particularly ketogenic diets, may provide benefit as a therapeutic or preventive strategy in cancer, alone or as an adjunct to pharmacology. The argument derives from several points of evidence: There is a close association between cancer and both diabetes and obesity. Extensive evidence shows that low carbohydrate diets are the most effective dietary treatment of Type 2 diabetes and dietary adjunct in Type 1. Such diets also target all the markers of metabolic syndrome. Finally, de facto reduction in carbohydrate likely contributes to total dietary restriction, which is effective in the prevention and treatment of cancer. The idea is consistent with recent interest in treating cancer with drugs that target diabetes. To move forward, we must understand obesity and diabetes as response to a hyperglycemic state rather than simply a cause of downstream effects.

Electromyographic Record of Classical Conditioning of Eye Withdrawal in the Crab

Classical (Pavlovian) conditioning of the eye withdrawal reflex of the green crab, Carcinus maenas, was studied by recording electromyograms (EMGs) from the main abductor muscle of the eye (19a). The EMG record was a reliable indicator of the response, and it was always correlated with physical movement of the eye, whether evoked by the unconditioned stimulus (a puff of air to the eye), or by the conditioned stimulus (a mild vibration of the carapace). The EMG was used to study the acquisition of conditioned responses in animals with an immobilized eye. Six of eight experimental animals developed responses to the conditioned stimulus in a manner similar to that for animals with freely moving eyes; unpaired controls showed few responses. The results indicate that eye movement is not required for learning. Behavioral tests after conditioning and after the eyes had been freed supported this conclusion. The results exclude theories of classical conditioning of eye withdrawal that invoke a role for stimuli due to eye movement (such as a change in visual field).

Classical Conditioning in the Crab

The green crab, Carduus maenas, can be trained in a classical (Pavlovian) conditioning procedure. By pairing vibration on the carapace as a conditioned stimulus (CS) with air-puff to one of the eyes as an unconditioned stimulus (US), it is possible to teach the animals to retract the eye during CS presentations. Controls subjected to unpaired stimuli show lower rates of responding. Crabs can also be trained in a signalled avoidance procedure, but the similarity of acquisition curves to those for classical conditioning suggests that the mechanism may be Pavlovian. Electromyograms recorded from the eye muscles can be used to follow conditioning and show that animals can be conditioned with the eye restrained.

An Evolutionary and Mechanistic Perspective on Dietary Carbohydrate Restriction in Cancer Prevention

Author(s): Fine, Eugene J. | Abstract: The confluence of basic cell biochemistry, epidemiological and anthropologic evidence points to high dietary carbohydrate and the associated disruption of the glucose-insulin axis as causes of the current increase in metabolic disorders, metabolic syndrome, hypertension and cardiovascular disease. This hyperinsulinemic state likely contributes, as well, to an increased mutagenic microenvironment, with increased risk for cancer. This critical review discusses these risks in their historical and evolutionary context. The evidence supports the benefits of lowering the glycemic load of the diet as a preventive measure against the development of cancer.

FTIR spectroscopy and sequence prediction: Structure of human α2-macroglobulin

The structure of a plasma proteinase inhibitor α2-Macroglobulin (α2m) is determined by FTIR spectroscopy and a number of sequence-structure prediction algorithms. In addition, α2M dimers and complexes with methylamine and trypsin are examined. Our FTIR results estimate a helix content of 5–15% and a β-sheet content of 28–36%. None of the sequence prediction algorithms used in this study predicted values close to experimental data. Considerable differences in the FTIR spectra of α2M dimer are observed and somewhat smaller changes are seen upon reaction of α2M with methylamine and dithiodipyridine (DTP).

Operant Conditioning in the Crab

Several operant conditioning procedures have been successfully used with the green crab, Carcinus maenas. Animals learned to press a bar for food reward and showed high rates of responding on a continuous reinforcement schedule or on a fixed ratio 2 schedule (every other response reinforced). Animals on a variable time schedule showed lower rates of responding. Crabs readily learned a spatial discrimination task in which one bar causes food to be dispensed (S+) and one is inactive (S-). The eye elevation system of the green crab can also be used in a yoked procedure using air puff to the eye as punishment. Master animals reduced the number of eye elevations after reflex withdrawal and showed longer latency compared to yoked controls. A delay introduced between response and punishment reduced the suppressing effect of the punishment.