Random phosphorylation of the two heads of thymus myosin and the independent stimulation of their actin-activated ATPases

Like other vertebrate nonmuscle myosins, thymus myosin contains two phosphorylatable light chains. Phosphorylation of these light chains regulates the actin-activated ATPase of this myosin. The time courses for the phosphorylation of both monomeric and filamentous thymus myosin by gizzard myosin light chain kinase fitted single exponentials to greater than 85% phosphorylation. This indicates that the two heads of thymus myosin are phosphorylated at the same rate and suggests that these phosphorylations are random processes. The actin-activated ATPases of thymus myosins with different levels of light chain phosphorylation were also determined. A linear relationship was obtained between the extent of light chain phosphorylation and stimulation of the actin-activated ATPase. Since thymus myosin appears to be phosphorylated randomly, this linear relationship indicates that phosphorylation of one head of thymus myosin stimulates the actin-activated ATPase of that head independently of the phosphorylation of the second head. The apparent random phosphorylation of thymus myosin light chains contrasts with the reported ordered phosphorylation of the light chains of filamentous smooth (gizzard) muscle myosin. Also, while the actin-activated ATPases of the two heads of thymus myosin are regulated independently, both heads of gizzard myosin must be phosphorylated before the ATPase of either head is activated by actin.     

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.     

Ribulose bisphosphate carboxylase capacity and chlorophyll content in developing seedlings of Chenopodium rubrum L. growing under light of different qualities and fluence rates

In order to evaluate the aclimation of Chenopodium seedlings to different quantum fluence rates of R and BL, kinetics of Rubisco capacity, Chl content and chloroplast structure were studied. Under monochromatic light photoreceptors are stimulated selectively and their influence on biosynthetis capacities during chloroplast development can be studied.R irradiations saturate Rubisco capacity even at the lowest quantum fluence rates applied, whereas Chl a+b synthesis depends strongly upon fluence rate of R. Under BL irradiations, both Rubisco capacity and Chl content are fluence rate dependent. R irradiations favour Chl b synthesis relative to Chl a, whereas under BL Chl a content is high relative to Chl b. Under R irradiation Pfr is the main photoreceptor involved in regulation of Rubisco capacity whereas under BL a specific BL absorbing photoreceptor may control the response. From the fluence rate dependency under BL irradiations it is concluded that the blue region of the day light spectrum may be the sensor for monitoring fluence rate and causing the characteristic changes in shade and high/low WL adaptation with respect to Rubisco levels in Chenopodium.     

Effect of dopamine agonists or antagonists, TRH, stress and piglet removal on plasma prolactin concentrations in lactating gilts

The effect of dopamine agonists (ergocryptine), antagonists (chlorpromazine, haloperidol, reserpine, pimozide), thyrotropin releasing hormone or stress (restraint, piglet removal) on prolactin release was studied in primiparous lactating gilts. All animals were fitted with surgically implanted jugular catheters before farrowing. The only drug treatments which resulted in a significant change in PRL concentrations in blood were thyrotropin releasing hormone (increase) and ergocryptine (decrease). The results suggest that dopamine may not be the only regulator of prolactin in lactating pigs. Further studies are needed to identify drugs which would be useful in clinical situations for treatment of lactation failure due to low prolactin secretion. In the two stress-exposed groups, there was a gradual, steady decline in the plasma concentration of prolactin which resulted from loss of suckling contact with the piglets. Thus, snare restraint does not increase prolactin secretion in lactating sows confirming the results of other studies on pigs in different physiologic states.     

Efficacy of high-frequency ventilation in presence of extensive ventilation-perfusion mismatch

Ten anesthetized normal dogs were each given two methacholine inhalational challenges to produce large amounts of low ventilation-perfusion (VA/Q) regions but little shunt. After one challenge, high-frequency ventilation (HFV) was applied, whereas after the other conventional mechanical ventilation (MV) was used, the order being randomized. Levels of both ventilatory modes were selected prior to challenge so as to result in similar and normal mean airway pressures and arterial PCO2 levels during control conditions. Gas exchange was assessed by both respiratory and multiple inert-gas transfer. Comparing the effect of HFV and MV, no statistically significant differences were found for lung resistance, pulmonary hemodynamic indices, arterial and mixed venous PO2, expired-arterial PO2 differences, or inert-gas data expressed as retention-excretion differences. The only variables that were different were mean airway pressure (2 cm higher during HFV, P less than 0.04) and arterial PCO2 (10 Torr higher during HFV, P less than 0.002). These results suggest that in this canine model of lung disease characterized by large amounts of low VA/Q regions, HFV is no more effective in delivering fresh gas to such regions than is MV.     

Effects of transfusion-induced polycythemia on O2 transport during exercise in the dog

An increased hematocrit could enhance peripheral O2 transport during exercise by improving arterial O2 content. Conversely, it could reduce maximal delivery of O2 by limiting cardiac output during exercise or by limiting the distribution of blood flow to peripheral capillaries with high O2 extractions. We studied O2 transport at rest and during graded treadmill exercise in splenectomized tracheostomized dogs at normal hematocrit (38 +/- 3%), and 48 h after transfusion of type-matched donor cells. This procedure increased hematocrit (60 +/- 3%) but also increased blood volume (P less than 0.05). Following transfusion, resting cardiac output (QT) and heart rate were not different. During exercise, QT was significantly lower at each level of O2 consumption (VO2) at high hematocrit (P less than 0.01). A reduction in QT was also seen during polycythemic exercise with hypoxemia produced by breathing 12 or 10% O2 in N2. Despite the reduction in QT, mixed venous PO2 was not lower at high hematocrit, and the increase in base deficit with VO2 was not different from control measurements. O2 delivery (QT X arterial content) was similar at each level of VO2 at both levels of hematocrit, during both normoxic and hypoxic studies. Both systemic and pulmonary arterial pressures were increased at rest after transfusion (P less than 0.05). However, pulmonary and systemic pressures were not higher than control during exercise at high hematocrit. We conclude that a hematocrit of 60% with increased blood volume is not associated with a cardiac limitation of O2 delivery, nor does it interfere with peripheral O2 extraction during exercise in the dog.     

Role of hemoglobin P50 in O2 transport during normoxic and hypoxic exercise in the dog

High hemoglobin affinity for O2 [low PO2 at 50% saturation of hemoglobin (P50)] could degrade exercise performance in normoxia by lowering mean tissue PO2 but could enhance O2 transport in hypoxic exercise by increasing arterial O2 saturation. We measured O2 transport at rest and at graded levels of steady-state exercise in tracheostomized dogs with normal P50 (28.8 +/- 1.8 Torr) and again after P50 was lowered (19.5 +/- 0.7 Torr) by sodium cyanate infusions. Measurements were made during ventilation with room air (RA), 12% O2 in N2, or 10% O2 in N2. Cardiac output (QT) as a function of O2 consumption (VO2) was not altered by low P50 at any inspired O2 fraction (P greater than 0.05). With RA exercise, arterial content (CaO2) and O2 delivery (QT X CaO2) were unchanged at low P50, whereas mixed venous PO2 was reduced at each level of VO2. With exercise in hypoxia, CaO2 and O2 delivery were significantly improved at low P50 (P less than 0.05). Mixed venous PO2 was lower than control during 12% O2 (P less than 0.05) but not different from control during 10% O2 exercise at low P50. Despite a presumed decrease in tissue PO2 during RA and 12% O2 exercise, exercise performance and base excess decline were not significantly worse than control levels. We conclude that, in canine steady-state exercise, hemoglobin P50 is not an important determinant of tissue O2-extraction capacity during normoxia or moderate hypoxia. In extreme hypoxia, low P50 may help to maintain tissue PO2 by enhancing systemic O2 delivery at each level of QT.     

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.