Computer simulation of cellular movements: cell-sorting, cellular migration through a mass of cells and contact inhibition

The motility rules for cellular movement proposed earlier by Goel &; Rogers for engulfment of two or more intact embryonic tissues have been used to simulate on a computer the phenomena of cell-sorting, migration of individual cells through a mass of cells and contact inhibition of overlapping. These simulations in the most part are found to be consistent with the observations with real cells.     

Error catastrophe in and the evolution of the protein synthesizing machinery

The theory of the propagation of errors in the system of enzymes translating genetic information proposed earlier is developed further. It is shown that if initially for some of these enzymes the normal molecules are more than, for some less than, and for some equal to the erroneous ones, the evolution of the system can lead to many asymptotic conditions including error catastrophe and complete recovery from errors.     

The error catastrophe hypothesis with reference to aging and the evolution of the protein synthesizing machinery.

Recent investigations of the thermodynamics of protein denaturation, in particular of pressure effects, have questioned the fundamental importance, hitherto assumed, of hydrophobic interactions in the native conformations of proteins. The volume changes observed on protein denaturation are incompatible with the volume changes estimated on the basis of volume effects observed in low molecular weight model systems of the aliphatic groups. In the present paper the model systems generally considered are critically discussed. It is concluded, that solutions of low molecular weight alkanes may not be any adequate models of aliphatic groups in proteins. Studies of more appropriate model systems suggest that the volume changes to be expected, when buried aliphatic groups of proteins are exposed to water, are small and positive, and mainly due to structural changes of the water. These volume changes are in accordance with the volume changes actually measured of protein denaturation, and the latter volume effects are taken as supporting evidence of the importance of hydrophobic interactions in protein confonriations.     

On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.     

The information content of DNA and evolution

The information content of DNA molecules has been calculated for various organisms using experimentally measured codon frequencies as well as those estimated theoretically. A direct relationship between the information content and the evolutionary rank of the organism is observed. The biological and physical significance of information content in the context of evolution is discussed.     

On the computation of the tertiary structure of globular proteins II.

The semi-empirical approach proposed earlier (Y?as et al., 1978) to compute the tertiary structures of globular proteins is here amplified and developed further. Using, as input, information on sequence and certain averages of interatomic distances which can be semi-empirically estimated, structures have been computed for pancreatic trypsin inhibitor, lysozyme and staphylococcal nuclease which resemble those determined by X-ray diffraction methods. The approach used is compared and contrasted with others proposed recently.     

On the computation of the tertiary structure of globular proteins

A method is presented to compute the approximate locations of α carbon atoms of proteins using experimentally obtainable information. This information consists of distances between nearest neighbor α carbon atoms, locations of SS bonds, primary sequence of amino acids as reflected by hydrophobic and hydrophylic residues and the assumption of globularity. The method permits the reconstruction of structure similar to the real ones, and is readily extendable to compute structures more accurately by incorporating additional information.     

Cyanate from alkaline hydrolysis of cyanogen bromide-activated polysaccharides

Alkaline treatment (pH 12) of cyanogen bromide-activated Sepharose and Sephadex, both with or without coupled protein, released a thiol protease inactivator. It was successfully isolated by the combination of Sephadex G-10 gel filtration and Dowex 1-X2 ion-exchange chromatography. Infrared and chemical analysis show it to be the cyanate ion. Barbituric-pyridine colorimetric analysis, capacity test, and other evidence suggest that the carbamate group in the activated polysaccharides is the most likely precursor of the cyanate.     

Effect of quinolinate on aminotransferases

Quinolinate inhibits several aminotransferases (ornithine, alanine, and aspartate). However, it is considerably more potent as an inhibitor of liver and heart cytoplasmic aspartate aminotransferase. It is a much less potent inhibitor of mitochondrial aspartate aminotransferases. Quinolinate is bound to the active site of cytoplasmic aspartate aminotransferase. It has a much greater affinity for the pyridoximine-P than the pyridoxal-P form of the enzyme. According to kinetic results, the inhibition or dissociation constant of quinolinate is 0.2 and 20 mm, respectively, for the pyridoxamine-P and the pyridoxal-P forms of the enzyme. Since quinolinate is mainly bound to the pyridoxamine-P form: (a) it is a potent competitive inhibitor of α-ketoglutarate but has little effect when α-ketoglutarate is saturating even if the level of aspartate is low; (b) it decreases the effect of α-ketoglutarate on the absorption spectrum of the pyridoxamine-P form; and (c) it enhances the effect of glutamate on the absorption spectrum of the pyridoxal-P form. Quinolinate is also apparently bound to the apoenzyme since it inhibits reconstitution by either pyridoxamine-P or pyridoxal-P. Since quinolinate is a competitive inhibitor of α-ketoglutarate, it is possible that part of the inhibitory effect of quinolinate on hepatic gluconeogenesis could result from quinolinate inhibiting the conversion of aspartate to oxalacetate by the cytoplasmic aspartate aminotransferase. Quinolinate has no effect on either rat or bovine liver glutamate dehydrogenase or on kidney glutamate dehydrogenase.     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Analysis of double-helix motions with spin-labeled probes: binding geometry and the limit of torsional elasticity

The e.p.r.⁵ spectra of a family of spin-labeled probes non-covalently bound to DNA have been measured as functions of helix orientation, packing density and temperature. The spectra are interpreted in terms of the geometrical relations between the helix axis and the orbital containing the unpaired electron and in terms of the motions of the helix. Torsional and flexural motions can be distinguished.Spectra from well-ordered helices have been obtained using fully hydrated DNA fibers that are in thermodynamic equilibrium with unbound probe in dilute salt solution. The binding equilibria are similar to the equilibria in dilute DNA solution. The spatial relations between the spin label and the helix, inferred from the spectra, correspond closely to the structure expected on the basis of intercalation perpendicular to the helix axis and a sterically hindered amide bond between the spin label and the intercalating moiety of the probe. Viscometric measurements with one probe also indicate intercalation.Linear e.p.r. spectra of solutions, randomly condensed DNA, and fibers show substantial torsional motion but no detectable flexure on the linear e.p.r. time scale (> 300 ns). The correlation time of a propidium-based probe is much longer than that of aminoacridine intercalators. The probes with short correlation times are considered to be too weakly coupled to the adjacent base-pairs to be reliable indicators of DNA dynamics. For the propidium probe the correlation time, 30 nanoseconds, and its temperature dependence are compared with the properties expected according to four models: tight rotational coupling along the entire length of the helix; swivels at fixed intervals; a two-state exchange; and elastic rotational coupling between adjacent nucleotide pairs. In terms of the fourth model, the results suggest that each nucleotide pair undergoes random oscillation with an r.m.s. amplitude of not more than 4 ° to 5 ° at room temperature. That value agrees with estimates made in other ways.     

The identification of a serum viability factor for SV3T3 cells as biotin and its possible relationship to the maintenance of Krebs cycle activity

A low-molecular weight-factor (“Peak III”) from calf serum, which enhances the viability (and hence growth) of simian virus 40-transformed 3T3 (SV3T3), but not 3T3, cells when grown in low-serum (0.15–0.30%, $\text{v}\text{v}$) Dulbecco's modified Eagle's medium, has been identified as the vitamin biotin. The extraction procedure involved acidification of the serum to pH 4.5, boiling, ultrafiltration of the supernatant through a Pellicon membrane, and Sephadex G-25 chromatography. Peak III was identified as biotin for the following reasons. (i) The viability/growth activity was completely retained on an avidin-Sepharose but not a Sepharose column. (ii) Peak III preparations contained a compound which reacted with the cyclic ureide-specific reagent, p-dimethyl-aminocinnamaldehyde, and which migrated on thin-layer chromatography with the same R_f as biotin. (iii) Peak III and biotin had the same biological effects on SV3T3 cells, including a reduction in the number of dead cells, a lowering of the amount of lactic acid accumulated, and a synergism with iron in stimulating growth. (iv) They were not additive in their effects at saturating doses. To test the hypothesis that at least part of the biotin viability/growth effect may be due to the maintenance of Krebs cycle intermediate levels through the activation of pyruvate carboxylase, Krebs cycle intermediates were added singly to cells in low-serum medium without biotin. Malate, citrate, isocitrate, and fumarate (but not oxaloacetate, α-ketoglutarate, and succinate) were growth stimulatory for SV3T3 but not 3T3 cells. When added in combinations they were no more effective than alone.     

On the chemical structure of the apical droplet from eggs of Culex pipiens

The chemical nature of the apical droplet from eggs of Culex pipiens was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12β-hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14β-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with β-OH myristic acid.Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive ³²PO₄^?3 and ³⁵SO₄^?2 showed no significant incorporation of phosphorus, but a sulphur-containing anionic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as mono-and diglycerides. The structural evidence explains in part the surfactant properties of the apical drop.     

Penetrability of orally toxic protein from cobra venom through the gut of a blowfly

The oral toxicity of a radioiodinated toxic polypeptide isolated from a cobra snake venom as assayed by Sarcophaga falculata blowflies coupled with assays on competitive displacement have indicated that: (a) During 3–4 h 8% of the orally active toxin is able to pass through the digestive system of the fly; (b) the orally active toxin after passing the gut binds to body tissues. The strong affinity of the toxin to tissue membranes explains its absence in the insect's hemolymph following oral applications as well as injection.The removal of traces of phospholipase A, which is extremely toxic, by injection of the orally active toxin has significantly lowered its injection toxicity without affecting its oral toxicity, thus indicating the absence of any interaction with phospholipases in oral toxicity. This conclusion was supported by additional experimentation.     

An analysis of sandbathing and grooming in the kangaroo rat (Dipodomys merriami)

The sandbathing and grooming behaviour of ten kangaroo rats (Dipodomys merriami) were recorded on sand and woodchip substrates after periods of 0,1,5 and 10 days without sand. Sandbathing is restricted to the sandy substrate. Grooming occurs on both, but with a higher frequency on sand. Increases in both grooming and sandbathing occur with increasing sand deprivation, but the temporal patterning does not change. D. merriami tends to alternate sandbathing components in contrast to other Dipodomys species. Lipid on the pelage increases noticeably with sand deprivation and decreases during a sandbathing bout; sand appears to be removed from the pelage by shaking and grooming. These findings suggest a three-process system for care of the body surface.     

The influence of caging conditions and hormone treatments on fighting in male and female hamsters

After gonadectomy, more individually caged female hamsters fought prior to the initiation of hormone treatments than did group-caged females. Daily injections of testosterone propionate (TP), estradiol benzoate (EB), or progesterone (Prog) had no influence on the number of individually caged females that fought. However, TP and EB were effective in increasing the number of group-caged females that fought. In contrast to females, both individually and group-caged males fought infrequently after castration. Daily injections of TP, EB, or Prog were effective in increasing the number of individually caged males that fought, while only TP and EB were effective in group caged males. Prog failed to increase the number of group-caged hamsters of either sex that fought.     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.     

Effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on isolated lepidopteran mitochondria

An investigation was undertaken to determine the effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on mitochondria isolated from Bombyx mori midgut epithelium. Using manometric and colorimetric techniques, the investigation revealed that toxic polypeptides had stimulatory effects on mitochondria oxygen uptake and inhibitory effects on ATP production. These results indicated that B. thuringiensis δ-endotoxin could act as an uncoupler of oxidative phosphorylation. Loss of ATP production caused by the action of the δ-endotoxin would lead to metabolic imbalance and possible cell death.