Modification of spontaneous unit discharge in the lateral geniculate body by a magnetic field.

The effects of a 1230-G static magnetic field on spontaneous discharge frequency and discharge pattern of principle cells in the cat's lateral geniculate body (LGB) were examined. In 45% of cells studied, a decrease in frequency was seen after the field was turned on. This progressed, even after the field was turned off, with return to baseline after an average duration of 250 s. Onset typically was 75 s after the field was activated, with maximum effect occurring 135 sec thereafter. In 67% of those cells which exhibited a decrease in frequency and in 50% of those which did not, a change in discharge pattern, as reflected by the interspike interval histogram, was seen. When present, this was manifested as a decrease in short interspike intervals. The change in the interspike interval histogram usually persisted longer than the change in frequency. The gradual onset and prolonged time course of changes in LGB cell activity suggest either an alteration in the synaptic ionic environment or in neurotransmitter availability. It is hypothesized that strong magnetic fields produce a partial realignment of diamagnetically anisotropic molecules within the cell membrane, thereby distorting ion-specific channels sufficiently to alter their function.

Conformation of the metastasis-inhibiting laminin pentapeptide.

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formation in vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.

Conformational effects of amino acid substitutions at positions 10, 12, and 13 in the P21 protein.

Substitutions of amino acids for Gly 12 or Gly 13 in the ras oncogene-encoded P21 proteins have been demonstrated to produce unique structural changes in these proteins that correlate with their ability to produce cell transformation. For example, the P21 proteins with Arg 12 or Val 13 are both known to be actively transforming. Recent site-specific mutagenesis experiments on the transforming Arg 12 protein have found that the substitution of Val for Gly 10 has no effect on transforming activity whereas the substitution of Val for Gly 13 led to a loss of transforming activity. In this study, we examine the structural effects of these substitutions on the amino terminal hydrophobic decapeptide (Leu 6-Gly 15) of P21 using conformational energy analysis. The results show that the transforming proteins with Gly 10 and Arg 12 or Val 10 and Arg 12 can both adopt the putative malignancy-causing conformation, whereas, for the nontransforming protein with Arg 12 and Val 13, this conformation is energetically disallowed. These results further support the theory that due to structural changes the transforming P21 proteins are unable to bind to some regulatory cellular element which may be the recently identified binding protein responsible for the induction of increased GTPase activity in normal P21 compared with transforming mutants.

Conformational effects of the substitution of Arg for Gly 13 in the ras oncogene-encoded P21 protein.

The effect of the substitution of Arg for Gly 13 on the structure of the transforming region decapeptide (Leu 6-Gly 15) of the ras oncogene encoded P21 protein has been investigated using conformational energy analysis. A human malignancy has been identified that contains a ras gene with a single mutation in the thirteenth codon such that the encoded protein would have Arg substituted for Gly at this position, and transfection of cells in culture with this gene results in malignant transformation. Conformational analysis demonstrates that the Arg 13 decapeptide adopts a conformation identical to that for other peptides with substitutions at position 13 (Asp 13, Val 13) from transforming proteins that is distinctively different from that for peptides (Gly 13, Ser 13) from normal, nontransforming proteins. This is found to be an indirect effect resulting from changes in the conformation of Gly 12 produced by substitutions at position 13. These results are consistent with recent analysis of crystallographic data of proteins on conformational preferences for glycine in tripeptide sequences.

Structure of the carboxyl terminus of the RAS gene-encoded P21 proteins.

The three-dimensional structures of the carboxyl-terminal regions of the P21 protein products of the human Harvey (Ha), Kirsten (KiA and KiB), and neuroblastoma (N) RAS oncogenes and various mutants have been determined by using conformational energy analysis. The carboxyl-terminal region of P21 has been strongly implicated in the binding of the protein to the inner surface of the plasma membrane without which the protein is inactive. The only invariant residue in this region is Cys-186, which is necessary for the post-translational addition of palmitic acid. The surrounding sequences of the active native proteins differ considerably. Nevertheless, certain amino acid substitutions in this region are known to eliminate membrane binding and protein activity, suggesting that there is a conserved common structural feature in this region in the native proteins that is disrupted in the mutant proteins. Conformational energy analysis shows that the four native P21 proteins have a common structure in the form of an alpha-helix for the terminal pentapeptide. A mutant, pBW277, that fails to bind to the membrane and is inactive cannot adopt an alpha-helical structure in this region because of a proline at position 188. Another mutant, pBW766, that retains membrane binding and activity, on the other hand, retains the preference for an alpha-helical conformation in the terminal pentapeptide. These findings suggest that, despite various amino acid sequences in this region, the carboxyl-terminal pentapeptides of the P21 proteins form a distinctive structural domain that must have an alpha-helical structure for membrane binding and intracellular activity.

Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins.

The low-energy conformations for a series of peptides based on the sequence of the ras P21 protein from position 55 to position 67 have been computed using conformational energy analysis. These sequences differed at position 61 and contained Gln, Pro, Leu, Lys, and Arg at this position. P21 proteins with Gln, Glu, or Pro at this position do not cause cell transformation at normal levels of expression; proteins with substitutions of at least 14 other amino acids at this position (Leu, Lys, and Arg having been found in tumors in place of the normally occurring Gln-61) do cause malignant transformation of cells in culture. We find that the segments of residues 55-67 from the nontransforming proteins (Gln- or Pro-61) adopt a structure that is energetically unfavorable for the same segment with Leu, Lys, or Arg at position 61. The critical feature of this structure is an alpha-helix from residues 62 to 68. Residue 61 (Gln or Pro) adopts an extended conformation. On the other hand, the segment from transforming proteins can adopt two structures, one all alpha-helical from residue 61 to residue 68 and the other a less-regular, higher-energy structure. The segments from the normal protein can adopt the all alpha-helical structure, a finding that can explain the fact that elevated intracellular levels of the normal protein also cause cell transformation. The results of the calculations suggest that specific changes in the structure of this region can account for the oncogenic effect of the proteins in which substitutions occur.

On the biologically active structures of cholecystokinin, little gastrin, and enkephalin in the gastrointestinal system.

The biologically active conformations of a series of four peptides [four cholecystokinin (CCK)-related peptides and enkephalin] in their interactions with gastrointestinal receptors have been deduced using conformational computational analysis. The two peptides that interact exclusively with peripheral-type CCK receptors are the heptapeptide COOH-terminal fragment from CCK (CCK-7) and the analogous sequence from cerulein (CER-7) in which threonine replaces the methionine proximal to the NH2 terminus. The two peptides that interact exclusively with the gastrin receptor in the stomach are the active COOH-terminal fragment of little gastrin and the COOH-terminal tetrapeptide sequence common to all of these peptides, CCK-4. We find that preferred conformations for the peripherally active peptides CCK-7 and CER-7 are principally beta-bends, whereas little gastrin and CCK-4 are fundamentally helical. In the class of lowest energy structures for both CCK-7 and CER-7, the aromatic rings of the tyrosine and phenylalanine lie close to one another whereas the tryptophan indole ring points in the opposite direction. This structure is superimposable on the structures of a set of rigid indolyl benzodiazepine derivatives that interact with complete specificity and high affinity with peripheral CCK receptors further suggesting that the computed beta-bends are the biologically active conformation. The biologically active conformation for CCK-4 and the little gastrin hexapeptide has also been deduced. By excluding conformations common to CCK-7 and CCK-4, which do not bond to each other's receptors, and then by selecting conformations in common to CCK-4 and the gastrin-related hexapeptide, which do bind to each other's receptors, we deduce that the biologically active conformation at the gastrin receptor is partly helical and one in which the indole of tryptophan and the aromatic ring of phenylalanine are close to one another while the methionine and aspartic acid side chains point in the opposite direction. These major differences in preferred structures between the common CCK-7/CER-7 peptides and the common CCK-4/little gastrin peptides explain the mutually exclusive activities of these two sets of peptides. We have observed that [Met]enkephalin strongly antagonizes the action of the naturally occurring peripherally active CCK-8 (CCK-7 with an NH2-terminal aspartic acid residue added). The computed lowest energy structures for this opiate peptide closely resemble key features of the computed CCK-7/CER-7 structure, further supporting the proposed structure.

Magnetic field influence on central nervous system function.

The effects of strong static magnetic fields on the excitability of striate cortex in adult cats was studied. The visual evoked response was used as a measure of cortical excitability. In all animals a 1200-G field was associated with a significant decrease in both amplitude and variability of the evoked response. This effect began more than 50 s after the field was turned on and persisted, even after the field was turned off, for several minutes. This phenomenon appears to be due to action of the magnetic field at the synapse rather than on axonal conduction.

A limiting factor in the "normalization" of schizophrenic orienting response dysfunction.

Forty schizophrenic patients, 40 non-schizophrenic patients, and 40 normal subjects were given 60 each alternating 1000- and 2000-Hz, 1-second tones at 60 dB. Half of each sample, the Press Group (PG), had to press a pedal to the high (low) target tone, ignoring the nontarget tone. The other half, the Nonpress Groups (NPGs), were given no reason to attend. Skin conductance response (SCR), finger pulse volume (FPV), and electroencephalographic (EEG) activity were recorded. NPG schizophrenic subjects were more often nonresponsive in both SCR and FPV than other samples, but less often responsive in EEG only when a 20 percent criterion of alpha blockade was used. Schizophrenic subjects showed greater consistency of OR nonresponsiveness in SCR and FPV, and nonsignificantly greater consistency in criterion alpha block, pointing to a deficit in orienting response (OR) rather than in peripheral response. When the targeted signal was given, schizophrenic subjects showed the same response as other groups in all systems. This was not due to an indiscriminate increase in reactivity, since response increase centered on the targeted signal itself in all groups. As the target signal was repeated, autonomic OR in schizophrenics declined sharply so that they again became underresponsive. Thus, OR "normalization" achieved by targeting significant signals is restricted to relatively early responsiveness. The rapid decline in autonomic OR may help explain differences in schizophrenic subjects between P300 and autonomic ORs to significant stimuli. Schizophrenic subjects were no different from controls in bilateral SCR or FPV asymmetry, but displayed less frequent criterion alpha blockade and reduced background alpha power in the left hemisphere. Each system showed a different pattern of bilateral asymmetry, reflecting complex, not well understood relations among these responses. This was further emphasized by the fact that skin conductance level (SCL) incremented over trials in PG subjects, reflecting sustained activation, while EEG background showed an increase in slower wave power, consistent with reports of increased drowsiness. The only drug effect seen was a lowering of SCL. Neuroleptics were associated with a flexible inhibitory control of SCL, permitting normal-like increment when circumstances required. Depressed patients' data suggested they might show heightened OR nonresponsiveness to innocuous stimuli which might not be subject to "normalization" by manipulation of stimulus significance; hence OR deficit might still differentiate schizophrenic from depressive patients.

Bilateral skin conductance, finger pulse volume, and EEG orienting response to tones of differing intensities in chronic schizophrenics and controls.

Skin conductance (SCOR), finger pulse volume (FPV-OR), and EEG orienting responses were examined to repeated tones of either 60- or 90-dB intensity in chronic schizophrenics, nonschizophrenic psychiatric patients, and normals. SCOR reaffirmed previous findings with schizophrenics displaying significantly more frequent nonresponsiveness to 60-dB tones, and faster habituation among patients who did respond. Increased stimulus intensity decreased the incidence of nonresponsiveness to the level of controls, but did not alter the rapid habituation of schizophrenics. These results generalized fully to the FPV-OR, despite the independence demonstrated between SCOR and FPV-OR, but did not generalize to EEG response. There were no significant differences between schizophrenics and controls in EEG reactivity-only in background activity, particularly in a slowing of dominant alpha frequency in schizophrenics. Schizophrenics displayed the same degree of bilateral asymmetry as controls in both SCOR and EEG; there was no evidence of a specifically schizophrenic asymmetry. Schizophrenics nonresponsive in either SCOR or FPV-OR showed significantly greater Conceptual Disorganization and Emotional Withdrawal, and significantly less Excitement than responders in blind clinical ratings on the Brief Psychiatric Rating Scale. None of the findings could be attributed to the effect of neuroleptics. Comparisons between medicated and nonmedicated patients showed no drug-associated effect on any OR variable under study. Drug effects were apparent only in skin conductance level (SCL). Neuroleptics were associated with a significant reduction in SCL in both schizophrenics and nonschizophrenics, together with a flattening of an otherwise incrementing SCL among schizophrenics.

The lumi-aggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets.

We have developed a new instrument that simultaneously monitors aggregation and secretion of ATP from the same sample of platelets. Aggregation is determined by the usual turbidimetric method; secretion of ATP is followed by measuring luminescence from the firefly luciferase system at right angles to the aggregometer light path. Data are recorded on a two-pen strip chart recorder and reveal the temporal relations between the two platelet responses.