Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

Tryptamine, a Substrate for the Serotonin Transporter in Human Platelets, Modifies the Dissociation Kinetics of [3H]Imipramine Binding: Possible Allosteric Interaction

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Epidemiological trends of dermatophytoses and dermatophytes in Jerusalem between 1954 and 1981

Data for dermatophyte infections analysed for five 3-year periods between 1954 to 1981 led to the following conclusions: (1) Tinea pedis, tinea cruris and tinea manuum showed an increase in the 50's and 60's and declined in the 70's; (2) Tinea unguium and tinea corporis showed an increase during the whole period; (3) At all these sites, the percentage of Trichophyton rubrum, the main etiologic agent, increased steadily over the periods while the percentage of Trichophyton mentagrophytes, the secondary etiological agent, decreased. Epidermophyton floccosum, the third etiological agent in these sites, showed no sharp fluctuations; (4) These three dermatophytes which show similar microclimatic requirements and favour the same microecological niches, were called glabrohydrophilic. In tinea corporis they form a definite subset, their percentage being similar to that at other glabrous sites; (5) Tinea capitis was at its peak in the 50's, decreased sharply until the second half of the 70's, its main etiological agent being Trichophyton violaceum. Since 1979, an increase of tinea capitis occurred due to the newly introduced Microsporum canis; (6) Dermatophytes favouring scalp hair were called trichophilic. In tinea corporis they form a definite subset, their percentage being similar to that of tinea capitis; (7) A comparison with other studies from this country shows that macroclimate (i.e. humid warm coastal climate compared with dry cooler inland-mountain climate) is not an important factor in the etiology of tinea.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features are as important as sequence homologies inDrosophila heat shock gene upstream regions

Summary Pelham has shown that theDrosophila hsp 70 gene is not transcribed under heat shock conditions unless a given upstream region is present. Davidson et al. have recently compiled a list of sequences homologous to this region in otherDrosophila heat shock genes. They proposed that a set of unlinked genes, such as the heat shock genes, could be coordinately induced through an interaction in cis with a common regulatory molecule. That this interaction involves structural elements is suggested by the fact that these upstream regions share inverted repeats as well as areas of Z-DNA potential. Furthermore, using the Calladine-Dickerson rules for local helical parameters, we show that these regions share structural homology. This is significant because the presence of regions homologous to a derived consensus sequence does not necessarily imply structural similarity. Therefore, we suggest that these structural features are at least as important as the sequence homologies in enabling the heat shock response.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

Development of an improved medium for the isolation of 'Haemophilus somnus'

A medium containing lincomycin (3 μg/ml), cycloheximide (100 μg/ml) and chloral hydrate (0–1 %) was superior to all others examined for the isolation of 'Haemophilus somnus' from material contaminated with Proteus species.     

Ca2+ displacement by Polymyxin B from sarcolemma isolated by 'gas dissection' from cultured neonatal rat myocardial cells

Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from 'gas dissected' cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was 0.172 +/- 0.02 nmol/microgram membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or 0.072 +/- 0.01 nmol/microgram protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to 0.227 +/- 0.02 nmol/microgram protein (P less than 0.05), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes.