Biomedical subjects

I Marchand

Publications and source records attributed to I Marchand.

11 recordsLinked to original sources

PubMed2002

Quantification of subcellular glycogen in resting human muscle: granule size, number, and location.

A few qualitative investigations suggested that location of muscle glycogen (G) granules in specific sites may be associated with distinct metabolic roles. Similarly, it has been suggested that the acid-soluble and -insoluble G fractions (macro- and proglycogen, respectively) are different metabolic pools and also could exist as separate entities. We employed a transmission electron microscopic technique to quantify subcellular G particle size, number, and location in human vastus lateralis biopsies of 11 resting men. The intra- and interobserver variability for the various measures was generally <4%. Granule size and number were quantified in subcellular compartments (subsarcolemmal, intra- and intermyofibrillar). Subcellular location was critical: G was more densely concentrated in the subsarcolemmal than in the myofibrillar space, whereas the single-particle volume was greater in the latter. Single-particle diameter ranged from 10 to 44 etam and followed a continuous, normal distribution. This implies that proglycogen is not a distinct entity, but rather that pro- and macroglycogen are divisions of smaller and larger molecules. These results demonstrate a compartmentalized pattern of subcellular G deposition in human skeletal muscle for both the size and density of granules.

Adult↗

PubMed2001

High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements.

Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal conditions ('helper' function). Whereas the C-terminal one-third of the encoded protein participates in host transcription shutoff, the N-terminal two-thirds harbours a protein kinase ('PK') activity with broad specificity. However, how this activity relates to helper function is unclear. Here, a truncated gene 0.7 encoding PK was fused to an IPTG-inducible T7 late promoter and to a translation initiation region from a T7 late gene, and inserted into the chromosome of an E. coli strain expressing T7 RNA polymerase. After induction, total protein synthesis remains unchanged but with over 40% devoted to PK synthesis, an amazing figure for the expression of a single-copy gene. Mutations abolishing PK activity reduce this expression by 3-fold. Thus, PK activity stimulates PK expression when the latter is controlled by T7 late genetic elements. Further experiments show that stimulation occurs at both transcriptional and post-transcriptional levels. The helper function may therefore correspond to a PK-mediated stimulation of late expression, the mechanism of which is discussed. The possibility of exploiting the PK activity for improving E. coli expression systems is also considered.

Bacteriophage T7↗

PubMed2001

Bacteriophage T7 protein kinase phosphorylates RNase E and stabilizes mRNAs synthesized by T7 RNA polymerase.

The T7 protein encoded by the early gene 0.7 exhibits bifunctional activity. Whereas its C-terminal one-third participates in host transcription shut-off, the N-terminal two-thirds bears a protein kinase ('PK') activity that can phosphorylate a number of host proteins in addition to itself. Here, we show that, when PK is expressed in uninfected Escherichia coli cells, the C-terminal half of RNase E and the associated RNA helicase RhlB are heavily phosphorylated. Meanwhile, a subset of RNase E substrates, including the lac and cat mRNAs synthesized by bacteriophage T7 RNA polymerase (RNAP), are stabilized. These mRNAs are genuinely less stable than their counterparts synthesized by E. coli RNAP, because T7 RNAP outpaces translating ribosomes, creating naked, RNase E-sensitive mRNA stretches behind itself. Thus, PK alleviates this effect of desynchronizing transcription and translation. The relationship between the modification of RNase E and RhlB and these mRNA stabilization effects, which may be relevant to the stability of late T7 mRNAs during infection, is discussed.

DNA-Directed RNA Polymerases↗

PubMed2001

Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates.

The Escherichia coli endonuclease RNase E plays a key role in rRNA maturation and mRNA decay. In particular, it controls the decay of its own mRNA by cleaving it within the 5'-untranslated region (UTR), thereby autoregulating its synthesis. Here, we report that, when the synthesis of an RNase E substrate is artificially induced to high levels in vivo, both the rne mRNA concentration and RNase E synthesis increase abruptly and then decrease to a steady-state level that remains higher than in the absence of induction. Using rne-lacZ fusions that retain or lack the rne 5'UTR, we show that these variations reflect a transient mRNA stabilization mediated by the rne 5'UTR. Finally, by putting RNase E synthesis under the control of an IPTG-controlled promoter, we show that a similar, rne 5'UTR-mediated mRNA stabilization can result from a shortage of RNase E. We conclude that the burst in substrate synthesis has titrated RNase E, stabilizing the rne mRNA by protecting its 5'UTR. However, this stabilization is self-correcting, because it allows the RNase E pool to expand until its mRNA is destabilized again. Thus, autoregulation allows RNase E to adjust its synthesis to that of its substrates, a behaviour that may be common among autoregulated proteins. Incidentally, this adjustment cannot occur when translation is blocked, and we argue that the global mRNA stabilization observed under these conditions originates in part from this defect.

5' Untranslated Regions↗

PubMed2001

Gender differences in temperature and vascular characteristics during exercise recovery.

Temperature and vascular responses during exercise recovery were examined in men and women of similar age and fitness status (VO2max: 76 +/- 5 vs 73 +/- 5 mL O2 / kg Fat Free Mass x min). Forearm blood flow (venous occlusion plethysmography; FBF), rectal (Trectal) and forearm skin (Tskin) temperatures (degree C) were measured before and every 15 min up to 105 min (t105) during recovery from a 45-min run at 75% of VO2max. Results indicate Trectal decreased to pre-exercise levels within 25 min in men but reached and remained at values lower than baseline between 60 and 105 min of recovery in women. From 90 to 105 min of recovery, Tskin was lower in women than men (t105 : 29.0 +/- 1.3 vs 30.7 +/- 1.5; p <.05). Recovery FBF (mL/100mL x min) was higher in men than women from the start (6.2 +/- 1.9 vs 4.9 +/- 1.9) to the end of recovery (t105 = 1.7 +/- 0.6 vs 2.6 +/- 1.1) (p <.05). Heat flux calculated at the forearm was higher in women and increased throughout the last hour of recovery (p <.05). Further investigations are needed to examine mechanisms underlying failure of post-exercise core and skin temperatures in women to stabilize at pre-exercise levels.

Adult↗

PubMed2001

Pro- and macroglycogenolysis: relationship with exercise intensity and duration.

We examined the net catabolism of two pools of glycogen, proglycogen (PG) and macroglycogen (MG), in human skeletal muscle during exercise. Male subjects (n = 21) were assigned to one of three groups. Group 1 exercised 45 min at 70% maximal O(2) uptake (VO(2 max)) and had muscle biopsies at rest, 15 min, and 45 min. Group 2 exercised at 85% VO(2 max) to exhaustion (45.4 +/- 3.4 min) and had biopsies at rest, 10 min, and exhaustion. Group 3 performed three 3-min bouts of exercise at 100% VO(2 max) separated by 6 min of rest. Biopsies were taken at rest and after each bout. Group 1 had small MG and PG net glycogenolysis rates (ranging from 3.8 +/- 1.0 to 2.4 +/- 0.6 mmol glucosyl units. kg(-1). min(-1)) that did not change over time. In group 2, the MG glycogenolysis rate remained low and unchanged over time, whereas the PG rate was initially elevated (11.3 +/- 2.3 mmol glucosyl units. kg(-1). min(-1)) and declined (P < or = 0.05) with time. During the first 10 min, PG concentration ([PG]) declined (P < or = 0.05), whereas MG concentration ([MG]) did not. Similarly, in group 3, in both the first and the second bouts of exercise [PG] declined (P < or = 0.05) and [MG] did not, although by the end of the second exercise period the [MG] was lower (P < or = 0.05) than the rest level. The net catabolic rates for PG in the first two exercises were 22.6 +/- 6.8 and 21.8 +/- 8.2 mmol glucosyl units. kg(-1). min(-1), whereas the corresponding values for MG were 17.6 +/- 6.0 and 10.8 +/- 5.6. The MG pool appeared to be more resistant to mobilization, and, when activated, its catabolism was inhibited more rapidly than that of PG. This suggests that the metabolic regulation of the two pools must be different.

Adult↗

PubMed2001

Pro- and macroglycogenolysis during repeated exercise: roles of glycogen content and phosphorylase activation.

This study examined the relationship between preexercise muscle glycogen content and glycogen utilization in two physiological pools, pro- (PG) and macroglycogen (MG). Male subjects (n = 6) completed an exercise and dietary protocol before the experiment that resulted in one leg with high glycogen (HL) and one with low glycogen (LL). Preexercise PG levels were 312 +/- 29 and 208 +/- 31 glucosyl units/kg dry wt (dw) (P < or = 0.05) in the HL and LL, respectively, and the corresponding values for MG were 125 +/- 37 and 89 +/- 43 mmol glucosyl units/kg dw (P < or = 0.05). Subjects then performed two 90-s exercise bouts at 130% maximal oxygen uptake separated by a 10-min rest period. Biopsies were obtained at rest and after each exercise bout. Preexercise glycogen concentration was correlated to net glycogenolysis for both PG and MG for bout 1 and bouts 1 and 2 (r < or = 0.60). In bout 1, there was no difference in the rate of PG or MG catabolism between HL and LL despite a 26% increase (P < or = 0.05) in glycogen phosphorylase transformation (phos a %) in the HL. In the second bout, more PG was catabolized in the HL vs. LL (38 +/- 9 vs. 9 +/- 6 mmol glucosyl units. kg dw(-1). min(-1)) (P < or = 0.05) with no difference between legs in phos a %. phos a % was increased in HL vs. LL but does not necessarily increase glycogenolysis in either PG or MG. Despite both legs performing the same exercise and having identical metabolic demands, the HL catabolized 2.3 (P < or = 0.05) times more PG and 1.5 (P < or = 0.05) times more MG vs. LL in bouts 1 and 2, indicating that preexercise glycogen concentration is a regulator of glycogenolysis.

Adult↗

PubMed2000

Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration.

The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by alpha-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P < 0.05) between HL and LL for total glycogen (438.0 +/- 69.5 vs. 305.7 +/- 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 +/- 38.1 vs. 227.3 +/- 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P < 0.001) was observed. Similar positive correlations (P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle.

Adult↗

PubMed1999

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo.

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.

Bacterial Proteins↗

PubMed1999

Gender differences in cardiovascular and catecholamine responses to cold-air exposure at rest.

Metabolic and cardiovascular responses were examined in 8 men and 9 women during 2 hrs of sitting at rest in 5 degrees C. The men and women had similar body fatness and both groups responded with similar changes relative to control in cardiac output, blood pressure, total peripheral resistance, VO2, respiratory exchange ratio, and rectal temperature. However, greater increases in stroke volume and decreases in heart rate and skin temperature were observed in men compared to women (p < 0.05). The contribution of CHO and fat to the total energy expended differed (p < 0.05) between genders; the CHO:fat ratio was 47:53 in men and 36:64 in women. No gender differences could be observed in plasma catecholamines. It is concluded that there are striking cardiovascular and metabolic gender differences in response to cold stress which cannot be accounted for by body fatness or catecholamine responses.

Adrenergic alpha-Agonists↗

PubMed1998

Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection.

Translation inhibitors such as chloramphenicol in prokaryotes or cycloheximide in eukaryotes stabilize many or most cellular mRNAs. In Escherichia coli, this stabilization is ascribed generally to the shielding of mRNAs by stalled ribosomes. To evaluate this interpretation, we examine here how inhibitors affect the stabilities of two untranslated RNAs, i.e., an engineered lacZ mRNA lacking a ribosome binding site, and a small regulatory RNA, RNAI. Whether they block elongation or initiation, all translation inhibitors tested stabilized these RNAs, indicating that stabilization does not necessarily reflect changes in packing or activity of translating ribosomes. Moreover, both the initial RNase E-dependent cleavage of RNAI and lacZ mRNA and the subsequent attack of RNAI by polynucleotide phosphorylase and poly(A)-polymerase were slowed. Among various possible mechanisms for this stabilization, we discuss in particular a passive model. When translation is blocked, rRNA synthesis is known to increase severalfold and rRNA becomes unstable. Meanwhile, the pools of RNase E and polynucleotide phosphorylase, which, in growing cells, are limited because these RNases autoregulate their own synthesis, cannot expand. The processing/degradation of newly synthesized rRNA would then titrate these RNases, causing bulk mRNA stabilization.

Chloramphenicol↗