Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.     

Effect of acute changes in the PaCO2 on acid-base parameters in normal dogs and dogs with metabolic acidosis or alkalosis

There is a linear relationship between the PaCO2 and blood hydrogen ion concentration in normal dogs, but for theoretical reasons to be discussed, we questioned whether this relationship would apply in animals with metabolic acidosis or alkalosis. To study this in more detail, animals were divided into three groups: normal, metabolically acidotic, and metabolically alkalotic. Following anesthesia and bilateral ureteral ligation, dogs were intubated and ventilated to produce acute steady state PaCO2 values corresponding to the range observed during disease states. Changes in the volume and electrolyte composition of the gastrointestinal fluid and urine as well as the concentration and distribution of lactate were evaluated in all experiments. We observed the previously described linear relationship between the PaCO2 and blood hydrogen ion concentration in normal dogs, but the slope of the regression line differed significantly from those of dogs with metabolic acidosis and metabolic alkalosis. On the other hand, there was a consistent relationship between the ratio of the PaCO2 values, but not the absolute PaCO2, and the change in the plasma bicarbonate concentration over a wide range of PaCO2 values in all groups of dogs. The chemical basis for these observations will be discussed.     

Gabamimetic properties of anxiolytic drugs

Diazepam (5 mg/kg, ip) and tracazolate (40 mg/kg, ip), a nonbenzodiazepine anxiolytic, blocked electrically-induced head-turning without producing sedation. Bicuculline and picrotoxin, GABA antagonists, at doses not affecting head-turning (2 mg/kg, ip) antagonized the effects of diazepam and tracazolate on head-turning. However, at the same dose, bicuculline was more effective as an antagonist of diazepam whereas picrotoxin was more effective as an antagonist of tracazolate. These results suggest that benzodiazepine as well as nonbenzodiazepine anxiolytics possess GABAmimetic activity. The difference in potency between bicuculline and picrotoxin as antagonists of diazepam and tracazolate may be related to their reported differences as GABA antagonists (e.g., site of receptor interaction).     

Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts

Depletion of intracellular potassium (K+) caused a marked reduction in the rate of endocytosis of receptor-bound low density lipoprotein (LDL) and epidermal growth factor (EGF) in human fibroblasts. K+ could be depleted slowly by a 3-hr incubation of cells in isotonic K+-free buffer. Rapid K+ depletion was induced by incubation of cells for 5 min with hypotonic medium, followed by transfer to isotonic K+-free buffer. Within 30 min of this treatment, cellular K+ levels fell by more than 60%. When the K+ level fell below a threshold of 40% of normal, the number of coated pits declined by 80% and the rate of endocytosis of 125I-LDL decreased by 70 to 95% despite normal to increased receptor binding. Similar results were obtained with 125I-epidermal growth factor. Addition of KCl to the culture medium up to 2 hr after K+ depletion restored cellular K+ levels and returned endocytosis of 125I-LDL promptly to normal. RbCl was as effective as KCl, but CsCl, LiCl, and (CH3)4NCl had no effect. Restoration by KCl was blocked by ouabain, indicating that uptake via the Na+/K+ ATPase was required. These data demonstrate that depletion of intracellular K+ reversibly arrests coated pit formation and receptor-mediated endocytosis in human fibroblasts.     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Isolation and characterization of a family of alpha-D-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells

A family of glycopeptides that contain nonreducing terminal alpha-D-galactosyl residues has been isolated from Pronase digests of delipidated Ehrlich ascites tumor cells. The glycopeptides, which comprise 17.2% of the total plasma membrane hexose, have an average molecular weight of 7500 and are precipitated by Griffonia simplicifolia B4 isolectin, wheat germ agglutinin, and Ricinus communis lectin. Exo- and endoglycosidase digestion, periodate oxidation, permethylation analysis, and lectin reactivity provided evidence for a tentative carbohydrate structure for the glycopeptide mixture. The glycopeptides possess tetraantennary branched structures containing a trimannosyl core N-glycosidically linked via an N,N'-diacetylchitobiosyl unit to an asparagine residue. Each branch contained repeating leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units resulting in a keratan-like structure, terminated with alpha-D-Galp-(1 leads to 3)-[alpha-D-Galp-(1 leads to 6)]-beta-D-Galp-units. The variation in the molecular weight observed for the glycopeptide mixture can be attributed to the variable amounts of leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units found in the branch chains.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Development of catecholaminergic phenotypic characters in the mouse locus coeruleus in vivo and in culture

While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. To approach this problem, we initiated parallel in vivo and in vitro studies of the mouse locus coeruleus (l.c.), a brainstem noradrenergic nucleus. The catecholaminergic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were used to monitor phenotype expression and development. TH catalytic activity and immunocytochemical reactivity were initially detectable on gestational Day 13 (E-13) in vivo, and adult levels of activity were approximately by the third postnatal week. Immunotitration studies indicated that the developmental increase was due to accumulation of enzyme molecules and not enzyme activation. The in vivo developmental profile of DBH approximated that of TH. To begin defining regulatory mechanisms, explants of embryonic brainstem were placed in culture. Explantation on E-12, prior to expression of TH or DBH, resulted in the de novo appearance of these phenotypic characters after 4 days. Explantation on E-18, after the enzymes are already expressed, was followed by a striking sixfold rise in TH activity. Immunotitration studies revealed that the increase in TH activity in E-18 cultures was attributable to increased molecule number, reproducing the in vivo results. Moreover, the E-18 explants, cultured for 3 weeks, attained higher plateau levels of TH activity than E-12 cultures, and this differences was due to increased molecule number. Morphometric analysis indicated that 3-week E-12 cultures actually had more l.c. cells than E-18 cultures, indicating that differences in TH were not due to increased cells in the E-18 l.c. Finally, systemic study revealed that the development of TH activity in culture increased progressively from E-11 to E-12 to E-13, suggesting that critical regulatory events occur at this time. Our studies suggest that the l.c. is an excellent model for the study of brain development in vivo and in vitro. Initial phenotypic expression and dramatic development occur in culture in the absence of normal targets, normal afferent innervation and, presumably, normal humoural milieu.     

Natural selection under population regulation

The dynamical behavior of multi-allele, one-locus systems is analyzed under population regulation. Weak selection is assumed. It is shown that for sufficiently large times, t, the nth time derivative of the population number N(t) is of order n}+1 in the selection coefficients. These order relations imply there is an asymptotic “quasi-equilibrium” in which population size and mean fitness change slowly relative to changes in gene frequencies. Consistent with the results of other authors, in quasi-equilibrium the mean fitness is second-order in the selection coefficients. In an effort to understand dynamic behavior beyond the immediate neighborhood of equilibrium, the topology of mean fitness surfaces is explored. In general, population regulation leads to regions of decreasing mean fitness in which there are important changes in gene frequencies. To illustrate this and other related phenomena, I analyze models in which there is logarithmic population control, and in which genotypic fitnesses W_i(x) are linear in the allele frequencies x. Exact solutions for mean fitness W(x) are obtained for two- and three-allele systems with symmetric fertilities and mortalities.