Changes in cytoplasmic carbohydrate content during Helleborus pollen presentation

Pollen grains of Helleborus foetidus and H. bocconei were exposed to low temperature treatments to simulate the natural events in pollen presentation of these two winter flowering species and to analyze the pollen carbohydrate content (glucose, fructose, sucrose and starch). In both species, cytoplasmic polysaccharides, monosaccharides and sucrose were found, while only Helleborus foetidus contained starch. Polysaccharide, sucrose and monosaccharide content varied as low temperature exposure time varied, a decrease in temperature decreases polysaccharide content and increases sucrose and monosaccharides. The relative quantities of the various types of carbohydrates were not affected by variations in the naturally occurring thermal cycles. Treatments did not greatly affect pollen viability. Although the occurrence of carbohydrates in pollen is known since many years, their function is still unclear. The findings of this research suggest a role of cytoplasmic pollen carbohydrates in resistance to low temperature exposure. The inter-conversion of carbohydrate type may be an adaptation for sustaining viability during pollen presentation that is particularly important for a winter flowering species such as Helleborus foetidus and H. bocconei .     

Glycogen content has no effect on skeletal muscle glycogenolysis during short-term tetanic stimulation

The effect of skeletal muscle glycogen content on in situ glycogenolysis during short-term tetanic electrical stimulation was examined. Rats were randomly assigned to one of three conditions: normal (N, stimulated only), supercompensated (S, stimulated 21 h after a 3-h swim), and fasted (F, stimulated after a 20-h fast). Before stimulation, glycogen contents in the white (WG) and red gastrocnemius (RG) and soleus (SOL) muscles were increased by 13-25% in S and decreased by 15-27% in F compared with N. Hindlimb blood flow was occluded 60 s before stimulation to produce a predominantly anaerobic environment. Muscles were stimulated with trains of supramaximal impulses (100 ms at 80 Hz) at a rate of 1 Hz for 60 s. Muscle glycogenolysis was measured from the decrease in glycogen content and estimated from the accumulation of glycolytic intermediates in the closed system. The resting glycogen content had no effect on measured or estimated glycogenolysis in all muscles studied. Average glycogenolysis in the WG, RG, and SOL muscles was 98.4 +/- 4.3, 60.9 +/- 4.0, and 11.2 +/- 3.6 mumol glucosyl U/g dry muscle, respectively. Hindlimb tension production was similar across conditions. The results suggest that in vivo glycogen phosphorylase activity in skeletal muscle is not regulated by the content of its substrate glycogen (range 80-165 mumol/g) during short-term tetanic stimulation in an anaerobic environment.     

Anaerobic energy provision in aged skeletal muscle during tetanic stimulation

The effect of age on skeletal muscle anaerobic energy metabolism was investigated in adult (11 mo) and aged (25 mo) Fischer 344 rats. Hindlimb skeletal muscles innervated by the sciatic nerve were stimulated to contract with trains of supramaximal impulses (100 ms, 80 Hz) at a train rate of 1 Hz for 60 s, with an occluded circulation. Soleus, plantaris, and red and white gastrocnemius (WG) were sampled from control and stimulated limbs. All muscle masses were reduced with age (9-13%). Peak isometric tensions, normalized per gram of wet muscle, were lower throughout the stimulation in the aged animals (28%). The potential for anaerobic ATP provision was unaltered with age in all muscles, because resting high-energy phosphates and glycogen contents were similar to adult values. Anaerobic ATP provision during stimulation was unaltered by aging in soleus, plantaris, and red gastrocnemius muscles. In the WG, containing mainly fast glycolytic (FG) fibers, ATP and phosphocreatine contents were depleted less in aged muscle. In situ glycogenolysis and glycolysis were 90.0 +/- 4.8 and 69.3 +/- 2.6 mumol/g dry muscle (dm) in adult WG and reduced to 62.3 +/- 6.9 and 51.5 +/- 5.5 mumol/g dm, respectively, in aged WG. Consequently, total anaerobic ATP provision was lower in aged WG (224.5 +/- 20.9 mumol/g dm) vs. adult (292.6 +/- 7.6 mumol/g dm) WG muscle. In summary, the decreased tetanic tension production in aged animals was associated with a decreased anaerobic energy production in FG fibers. Reduced high-energy phosphate use and a greater energy charge potential after stimulation suggested that the energy demand was reduced in aged FG fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous triacylglycerol utilization by rat skeletal muscle during tetanic stimulation

An isolated perfused rat hindquarter preparation was used to examine the utilization of endogenous triacylglycerol (TG) during 20 min of electrical stimulation. The sciatic nerve was stimulated with maximal tetanic trains at 0.5 Hz. The isometric tension generated by the gastrocnemius-plantaris-soleus muscle group was recorded, and muscle samples were taken pre- and poststimulation. Twenty minutes of stimulation significantly reduced endogenous TG from 6.78 +/- 0.84 to 4.64 +/- 0.64 mumol X g dry wt-1 (32%) in the red gastrocnemius muscle and from 7.70 +/- 0.61 to 6.66 +/- 0.80 mumol X g dry wt-1 (13.5%) in the plantaris muscle. Although TG content decreased by 16% in the soleus (28.2 +/- 5.0 to 23.8 +/- 4.4 mumol X g-1), the change was not significant. Stimulation had no effect on white gastrocnemius TG concentration (6.84 +/- 1.22 to 6.25 +/- 1.41 mumol X g-1). Thus oxidation of TG occurred primarily in muscles with a large proportion of fast-twitch oxidative-glycolytic fibers. Calculations from measurements of muscle energy stores and fuel uptake indicated that up to 62% of the aerobic energy was provided by endogenous TG. Carbohydrate oxidation contributed up to 28% and the remaining 10% may be accounted for by the oxidation of exogenous free fatty acids originating in the perfusate or from hindquarter adipose tissue. The magnitude of the fall in TG concentration in a given muscle was inversely related to the fall in glycogen concentration.     

Metabolic adaptation in phosphorylase kinase deficiency. Changes in metabolite concentrations during tetanic stimulation of mouse leg muscles.

1. Glycogen, nucleotides and glycolytic intermediates and products were measured before and during tetanus in the hamstrings-muscle groups of normal (C3H) and phosphorylase kinase-deficient (ICR/IAn) mice. 2. Phosphorylase kinase-deficient muscles contained 3-4-fold more glycogen and sustained a larger (approx. 2-fold), more rapid (11 +/- 2 ng/s faster) and more prolonged glycogenolysis during 120s tetanus despite their lack of phosphorylase a. 3. No significant change in total adenine nucleotide contents occurred during tetanus in either strain, but there was a 60-100-fold rise in IMP concentration to approx. 2mM in both strains. The initial rate of IMP formation was 6-fold more rapid (112 nmol/s per g) in phosphorylase kinase-deficient muscle. 4. Adenylosuccinate content rose to 36 nmol/g in phosphorylase kinase-deficient muscle and to 9 nmol/g in normal muscle at 45s tetanus, but then fell. 5. In phosphorylase kinase-deficient muscle, glucose 6-phosphate, a powerful phosphorylase inhibitor, was 56% of that in normal muscle. 6. The mass-action ratio of the phosphoglucomutase-catalysed reaction [glucose 6-phosphate]/[glucose 1-phosphate] was markedly lower than Keq. (approx. 17) in relaxed muscle of both strains (approx. 5-7), but rose significantly during tetanus to the value for Keq. 7. The data for IMP satisfy the criteria put forward by Rahim, Perrett & Griffiths [(1976) FEBS Lett. 69, 203-206] for a nucleotide activator of phosphorylase b: it should be present at a higher concentration in phosphorylase kinase-deficient muscle, its concentration should rise during muscle work, and it should attain a concentration comparable with its activation constant for phosphorylase b.     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Vagotomy effect on the content and synthesis of acetylcholine in autonomic ganglia in dog stomach

The effect of total, selective and highly selective vagotomy was studied on the content and synthesis of acetylcholine in the tissues of the gastric fundus and pyloric part of the stomach in mongrel dogs of either sex aged 3 to 6 years. Total vagotomy produced after 3 weeks a non-significant rise in the amount and a highly significant increase of the synthesis of ACh in the fundus, and a significant increase in the amount and synthesis of ACh in the pyloric part. Six weeks after the procedure the Ch content of the fundus and pyloric part decreased statistically significantly, while the synthesis rate was not different from the initial one. Three weeks after selective vagotomy the ACh content of the fundus and pyloric part was significantly increased. The rate of ACh synthesis was normal. Six weeks after vagotomy the content of ACh and its synthesis in the fundus were similar to those before vagotomy. In the same time the ACh synthesis rate was significantly decreased in the pyloric part, while the ACh content there was similar to the control value. Highly selective vagotomy with denervation of the fundus and corpus of the stomach but with maintenance of the innervation of the pyloric part caused after 3 weeks a rise of the ACh content of the fundus tissues. The ACh synthesis rate was not different from the normal one. In the pyloric part the ACh content was significantly decreased and the synthesis of ACh was somewhat inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the acetylcholine content of different brain regions of the rat during a learning experiment

Abstract— The ACh content of the rat hippocampus, visual cortex, auditory cortex and corpus striatum was estimated as so-called free, labile-bound and stable bound acetylcholine at different times after the learning of a brightness discrimination. In the hippocampal region the free acetylcholine was highly increased immediately after the training, whereas the stable bound ACh and the labile-bound fraction rose 70 min and 4 h respectively-after completion of training. Only small alterations of the ACh fractions were observed in the visual and auditory cortices. In the corpus striatum no changes appeared.     

Changes in RNA content in neurons of the superior olivary complex during monaural and binaural stimulation

The character of binaural competitive connections at the level of the superior olive was investigated cytospectrophotometrically in cats. As a result of monaural stimulation for 2 h or binaural stimulation for 1.5 h by rhythmic noise signal the RNA content in the neurons of the ipsilateral and contralateral medial and lateral nuclei increased significantly. The volume of functioning neurons in the nuclei studied either increased or remained the same as in the control. The increase in the RNA content in neurons of both the ipsilateral and contralateral medial and lateral nuclei suggests a uniform distribution of binaurally converging connections on the neurons of these nuclei. The results also suggest that the accumulation of cytoplasmic RNA takes place in response not only to excitation, but also to inhibition.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 10, No. 1, pp. 67–74, January–February, 1978.     

Changes in thoracic and right duct lymph flow and enzyme content during skeletal muscle stimulation.

In the pentobarbital-anaesthetized dog the effect of electrical stimulation of hindlimb skeletal muscles on thoracic and right duct lymph flow and enzyme content was examined. Increase in plasma creatine kinase (CK), L-aspartate-aminotransferase (AST) and lactic dehydrogenase (LDH) during 30-min muscle stimulation were not significantly altered by draining lymph. Both right duct and thoracic duct lymph flow trebled during stimulation. At the same time, the activity of the three enzymes examined decreased in right duct lymph and increased in thoracic duct lymph. Of the latter, only the increase in lymph CK was of a sufficient magnitude to have resulted in a detectable increase in plasma CK. CK was the smallest of the three enzymes studied and apparently preferentially entered the lymph, suggesting that the larger AST and LDH molecules were not likely to have entered the blood plasma directly from skeletal muscle. Rather their entry from some other tissue, possibly the formed elements of the blood, is indicated.     

Acetylcholine binding site in the vesicular acetylcholine transporter

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.