Platelet-derived growth factor. Specific binding to target cells

The binding of the human platelet-derived growth factor (PDGF) to Swiss mouse 3T3 cells have been investigated. The binding is specific and reversible. The dissociation constant is approximately 0.7 x 10(-9) M with approximately 400,000 binding sites/cell. Two forms of PDGF, PDGF I (Mr = 31,000) and PDGF II (Mr = 28,000), previously identified (Deuel, T. F., Huang, J. S., Proffitt, R. T., Baenziger, J. U., Chang, D., and Kennedy, B. B. (1981) J. Biol. Chem. 256, 8896-8899 and Deuel, T. F., Huang. J. S., Proffitt, R. T., Chang, D., and Kennedy, B. B. (1981) J. Supramol. Cell Biochem. 5 (Suppl.), 128) bind equally well to 3T3 cells. Polylysine and histone, but not cytochrome c, partially inhibit the binding of PDGF to 3T3 cells. Protamine sulfate blocks binding in a competitive manner and is capable of displacing PDGF previously bound to the cell surface. EDTA influenced neither the binding of PDGF to the cell surface nor the displacement of cell-bound PDGF. At 37 degrees C, PDGF bound to the cell surface is lost and iodotyrosine is released free into the supernatant, with each process having a t 1/2 of approximately 90 min. The binding activity of the putative PDGF receptor is markedly reduced by previous incubation with PDGF, thereby apparently regulating its activity in a manner similar to epidermal growth factor.     

Mendelian analysis of the organization of actin sequences in Physarum polycephalum

The genormic organization of the multiple actin DNA sequences in the lower eukaryote Physarum polycephalum was investigated by Mendelian mapping. Actin-homologous restriction endonuclease cleavage fragments detected by DNA blotting showed length polymorphisms when different strains were compared. These length polymorphisms were used as phenotypic markers for actin sequences in the genome. The meiotic assortment of the polymorphic restriction fragments was analysed, revealing four unlinked actin loci. The data for three-of the actin loci, ardB, C and D, are consistent with a single sequence or gene at each locus. The data for the other actin locus. ardA, is consistent with multiple linked actin sequences or genes.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S- containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8- 7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild- type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.     

Cerebral Synaptic Transmission During Anoxia Is Protected by Creatine

Synaptic transmission in cerebral tissue fails very rapidly in the absence of oxygen; the metabolic basis for this is not known. We report here that the transmission failure in the guinea pig hippocampal slice can be delayed threefold by exposing the tissue to extracellular creatine (Cr) for 3 h. The improved survival is associated with an increase of tissue phosphocreatine (PCr) concentration. These data argue that the metabolic basis for synaptic transmission failure is a fall in tissue ATP concentrations. They also indicate a way to protect brain tissue against anoxic damage.     

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.     

(Na+ + K+)-ATPase : phosphorylation-dependent cross-linking of the alpha-subunits in the presence of Ca2+ and o-phenanthroline

In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.     

Isolation of P700-chlorophyll-protein complex from a blue-green alga by a nondetergent method

A procedure has been developed for the isolation of a P700-chlorophyll-protein complex from a blue-green alga by a nondetergent method. The use of a Sepharose 4B column in conjunction with sucrose density-gradient centrifugation allows the separation of this complex from three other chlorophyll a-containing fractions which have higher ratios of carotenoids to chlorophyll a. Isoelectric focusing results in a single band with an isoelectric point at 4.5. Analysis of the major (green) fraction reveals the presence of P700. The absorption and emission spectra of this complex are reported.