Platelet activation studied by fluorescence polarization

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.     

The determination of the separation of tyrosine-99 and tyrosine-138 in calmodulin: Radiationless energy transfer

The average separation of the phenolic groups of tyrosine-99 and tyrosine-138 has been measured by radiationless energy transfer between each tyrosine and the nitro derivative of the second tyrosine. A separation of 16.7 ± 0.7 Å was found in the absence of Ca²⁺ and 15.5 ± 0.7 Å in the presence of Ca²⁺.     

Location of a binding site for 1-anilinonaphthalene-8-sulfonate on calmodulin

The quenching by radiationless energy transfer of the ultraviolet fluorescence of Tyr-99 and Tyr-138 by bound 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been employed to determine the separation of a hydrophobic binding site of 1,8-ANS from each of the tyrosines. The results suggest that the dominant binding site is located in the N-terminal region of domain III.     

Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay

The heads of mouse spermatozoa obtained 5 min after release from the excised caudae epididymides showed a characteristic fluorescence pattern in the presence of the fluorophore chlortetracycline (CTC). There was uniform fluorescence over the entire head with about half the sperm population showing a brighter line of fluorescence across the equatorial segment; this fluorescence pattern was designated “F.” After 90-min incubation in culture medium (CM) containing 2% (w/v) bovine serum albumin, most of the sperm heads showed a dark band of nonfluorescence over the equatorial and postequatorial segment, while the anterior portion of the head showed bright fluorescence. This fluorescence pattern was designated “B.” The time course for the disappearance of pattern F matched the time course of the appearance of pattern B, with a half-time of 30 min. The transformation was complete in 90 min. At longer times of incubation in CM, the percentage of spermatozoa showing pattern B declined; fluorescence over the entire head was lost, characteristic of the pattern for acrosome-reacted sperm (P. M. Saling and B. T. Storey (1979). J. Cell Biol.83, 544–555). Mouse sperm showing pattern B were able to undergo the acrosome reaction, either spontaneously or by induction with acid-solubilized zonae pellucidae from mouse eggs (H. M. Florman and B. T. Storey (1982). Dev. Biol.91, 121–130). The latter reaction was blocked by its specific inhibitor 3-quinuclidinyl benzilate (QNB). Mouse sperm showing pattern F could not be induced to undergo the acrosome reaction by exposure to solubilized zonae. This implies that the change from fluorescence pattern F to fluorescence pattern B corresponds with changes in the sperm which make them susceptible to undergo the acrosome reaction. This change occurs during the time interval previously determined to be needed for capacitation of mouse sperm in vitro in CM (M. Inoue and D. P. Wolf (1975). Biol. Reprod.13, 340–346). These results imply that spermatozoa showing CTC fluorescence pattern B can be considered to be capacitated and that a functional definition for capacitation is the acquired ability to undergo the acrosome reaction rapidly when treated with acid-solubilized zonae pellucidae. The CTC fluorescence assay provides for the first time a means to monitor the time course of epididymal mouse sperm capacitation in vitro.     

Effect of inhibitors of the lipoxygenase pathway on mouse myoblast fusion

In this study we examined the effects of inhibitors of the lipoxygenase and cyclooxygenase pathways on mouse myoblast fusion. The fusion of cloned mouse myoblasts was markedly inhibited, in a dose-dependent manner, when cells were cultured in medium supplemented with either phenidone (1-phenyl-3-pyrazolidione) or BW755c (3-amino-1-(3-tri-fluoromethylphenyl)-2-pyrazoline), drugs which have been reported to inhibit lipoxygenase and cyclo-oxygenase activities. Fusion was also inhibited when these cells were cultured in medium supplemented with esculetin (6,7-dihydroxycoumarin) which has been reported to inhibit lipoxygenase activity. Removal of the above inhibitors resulted in a return to control levels of fusion. Fusion was not demonstrably inhibited with aspirin (acetylsalicylic acid) and only inhibited to a minor extent with indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid); both of these drugs are inhibitors of cyclo-exygenase activity.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Characterization of temperate phages ofBacillus subtilis

Nine known temperature phages ofBacillus subtilis, including four that are newly isolated (ϱ6, ϱ10, ϱ14, and ϱ18), have been compared. Analysis by serology, immunity, host range, and adsorption site similarity place the phages into four groups: Group I, ϕ105, ϱ6, ϱ10, and ϱ14, which are 80–90% related; Group II, SPO2; Group III, ϕ3T and ϱ11, 100% related; and Group IV, SP16. The phage ϱ18 is largely uncharacterized, but is heteroimmune to other groups.     

An Automated Technique for the Radiographic Determination of Bone Age

We have developed an automated procedure for determining bone-age in the monkey, Macaca fascicularis. The status of epiphysial junctions and sesamoids were determined from radiographs. Epiphyses were scored as either open or closed, and sesamoids were scored as either present or absent. Bone-age was calculated by a computer program that stored and used a data base of probabilities for epiphysial closure and sesamoid appearance. The simplicity of scoring, combined with the speed and accuracy of the calculations, make this technique potentially adaptable for use in a variety of primate species.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Effect of constant and fluctuating temperatures on resting and active oxygen consumption of toads,Bufo boreas

Summary The relations of standard and active rates of oxygen consumption to body temperature (T_b) were tested in montane Bufo b. boreas and lowland Bufo boreas halophilus acclimated to constant T _b of 10, 20, or 30° C or to a fluctuating cycle of 5–30° C. Standard metabolic rates (SMR) of boreas acclimated to 30° C and halophilus acclimated to 10° C show pronounced regions of thermal independence but all other standard and active metabolic rates of groups acclimated to other thermal regimes are thermally sensitive. The SMR of both subspecies acclimated to the 5–30° C cycle are more thermally sensitive than those of similar individuals acclimated to constant T _b. In cases where the relation between SMR and T _b is linear for both halophilus and boreas at the same acclimation temperature, the slope and Q₁₀ of the relation for boreas are significantly higher than those of halophilus. Acclimation had little or no effect on the active metabolic rates of either subspecies. The relation between SMR and T _b of boreas maintained under field conditions (Carey, 1979) is matched only by those of individuals from the same population acclimated to 20° C.