Calmodulin supports the force-generating function in desensitized muscle fibers

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Separation of neutral reducing oligosaccharides derived from glycoproteins by HPLC on a hydroxylated polymeric support

A prototype hydroxylated polymeric support, Glyco-PAK N, was employed to separate a number of neutral reducing oligosaccharides derived from various glycoproteins by the hydrazinolysis/re-N-acetylation/mild acid hydrolysis procedure. General features of the separation properties of the matrix are described. A major advantage of the packing over silica-based supports is its stability, a property which is advantageous both in repetitive analytical chromatography and in the preparative isolation and spectroscopic identification of pure compounds, free of column breakdown products.     

Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (±1°C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45°C, the transition was at 7°C while for plants from the same clone, grown at 20°C, it was at −2°C. For mung bean and tomato the transition was between 9 and 12°C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.     

Effect of upper airway carbon dioxide on ventilation and blood gases in the awake pony

Carbon dioxide concentrations were increased during expiration in the upper one-half of the trachea, pharynx, and nasal sinuses to determine if elevation of upper airway CO2 would alter breathing or arterial blood gases in the awake pony. Carbon dioxide (100%) was injected into the midcervical trachea via a chronically implanted transcutaneous cannula during the first part of the animal's expiration. This maneuver elevated upper airway expiratory CO2 concentrations but prevented any exogenous CO2 from entering the lung and being absorbed into the arterial blood. Twelve experiments were performed on six ponies in which upper airway CO2 was elevated 2, 4, and 6% above the normal expired CO2 concentrations. Tidal volume increased in a dose dependent manner during upper airway CO2 exposure, but total ventilation was unchanged from base-line measurements made while the animal breathed room air. Arterial Po2 also increased during upper airway CO2 administration, reaching a mean value 6 Torr (1 Torr = 133.322 Pa) greater than the base-line values at the +6% CO2 exposure. We conclude that upper airway CO2 exposure alters breathing pattern slightly (increases tidal volume) and increases arterial PO2 in the awake pony.     

Preparation and biological activity of taxol acetates

Synthesis and biological activity of 2′-acetyltaxol and 7-acetyltaxol are reported. Activity is measured $\text{in}\text{vivo}$ by cytotoxicity toward the macrophage-like cell line J774.2, and $\text{in}\text{vitro}$ by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2′ results in loss of $\text{in}\text{vitro}$ activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on $\text{in}\text{vitro}$ microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for $\text{in}\text{vitro}$ activity and this position is available for structural modifications.     

Interaction of formamidines with octopamine-sensitive adenylate cyclase receptor in the nerve cord of Periplaneta americana L

Chlordimeform (CDM) and demethylchloridimeform (DCDM) mimic the action of octopamine in elevating adenylate cyclase activity in intact nerve cords of the American cockroach, Periplaneta americana. At a concentration of 1 x 10(-5)M, DCDM (13.5x increase within 20 minutes) is a more potent effector of the response than CDM (3x increase within 20 minutes), but both compounds show less efficacy than octopamine (23.5x increase within 15 minutes). DCDM also mimics the stimulatory effect of octopamine on adenylate cyclase activity in nerve cord homogenates whereas CDM has no demonstrable effect on this preparation. The octopamine- and DCDM-induced responses are competitively inhibited by phentolamine (1 x 10(-6)M) and cyproheptadine (1 x 10(-6)M) but not by propranolol (1 x 10(-6)M). DCDM and CDM inhibit the octopamine-induced activation of adenylate cyclase by 33% and 44% respectively. The results are discussed in light of the proposal that DCDM serves as a partial agonist and CDM as an antagonist of the octopamine receptor.     

Interaction of Pseudomonas Bacteriophage 2 with the Slime Polysaccharide and Lipopolysaccharide of Pseudomonas aeruginosa Strain BI

Purified slime polysaccharide B and lipopolysaccharide of Pseudomonas aeruginosa strain BI were shown to possess receptor-like properties in inactivating Pseudomonas phage 2, whereas lipoprotein and glycopeptide fractions were devoid of activity. On a weight basis, slime polysaccharide B was more effective than lipopolysaccharide in inactivating phage. The specificity of the reaction with slime polysaccharide B was indicated by the fact that slime polysaccharide A of P. aeruginosa strain EI failed to inactivate phage 2. Electron micrographs showed phage 2 in typical, tail-first position of attachment on intact cells of strain BI, slime polysaccharide B, and lipopolysaccharide. Tail fibers were discernible during phage attachment.