Synthesis time of beta-galactosidase in Escherichia coli B/r as a function of growth rate.

By analysing the kinetics of beta-galactosidase accumulation after induction, the synthesis time of beta-galactosidase in Escherichia coli B/r was found to be 75s in rapidly growing cells (1.36 and 2.1 doublings/h), and 90s in slowly growing cells (0.63 doubling/h). These values correspond to peptide-chain-elongation rates of 16 and 13 amino acids/s respectively, in agreement with previous findings, indicating that the peptide-chain growth rate is constant (presumably maximal) in fast-growing bacteria, but decreased in slowly growing bacteria [Forchhammer & Lindahl (1971) J. Mol. Biol. 55, 563-568].     

Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein.

The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.     

Differential rate of ribosomal protein synthesis in Escherichia coli B-r

The differential rate of ribosomal protein synthesis, α_r (ribosomal protein synthesis rate/total protein synthesis rate), was measured for Escherichia coli strain B/r growing at different steady-state rates ranging from 0.67 to 2.3 doublings/hour. For growth rates above 1.2 doublings/hour, α_r was found to be proportional to the growth rate μ (doublings/h), such that α_r = 0.09 μ, and the ribosome efficiency (amino acids polymerized/second per ribosome), calculated from α_r, was found to be 14 to 18 amino acids/second per ribosome. With decreasing growth rates below 1.2 doublings/hour, α_r was found to be increasingly greater than 0.09 μ and the ribosome efficiency gradually decreased such that at μ = 0.67, α_r = 0.085, and the ribosome efficiency was reduced by 30% and was equal to 10 to 13 ammo acids/second per ribosome. These results imply that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 × 10⁸ to 5 × 10⁸ amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10⁸ to 4 × 10⁸ amino acids/genome. These relationships were verified by direct measurements of the amounts of DNA, RNA and protein at different steady-state growth rates. In addition, protein accumulation was measured following a nutritional shift-up from succinate to glucose minimal medium. The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up.     

Peptide chain elongation rate and ribosomal activity in Saccharomyces cerevisiae as a function of the growth rate.

Summary The peptide-chain elongation rate of Saccharomyces cerevisiae at two different growth rates was estimated by the kinetics of radioactive labelling of nascent and finished polypeptides as described by Gausing, 1972, and Young and Bremer, 1976. The elongation rates of a diploid strain cultured in yeast nitrogen base supplemented with glucose or acetate were 9.3 amino acids/s and 5.5 amino acids/s at 30°C, respectively. These data together with published values on the ribosomal efficiency as a function of growth rate (Waldron and Lacroute, 1975) enable us to estimate the rate of synthesis of ribosomal proteins as a function of the rate of total protein synthesis, _r, and the fraction of ribosomes that are active in protein synthesis. We conclude that in S. cerevisiae _r, is largely independent of the growth rate while the fraction of active ribosomes decreases with decreasing growth rate.     

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.     

An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid.

Rates of protein synthesis in tissues can be calculated from the specific radioactivity of free and protein-bound amino acids at the end of a constant infusion of a labelled amino acid (Garlick, Millward & James (1973) Biochem. J. 136, 935--945]. The simplifying assumptions used in these calculations have been criticized [Madsen, Everett, Sparrow & Fowkes (1977) FEBS Lett. 79, 313--316]. A more detailed analysis using a programmable desk-top calculator is described, which shows that the errors introduced by the simplifying assumptions are small, particularly when the specific radioactivity of the free amino acid rises rapidly to a constant value.     

Evidence of heterogeneity of protein-turnover states in cultured cells.

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847--859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, 'chased' for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267--283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.     

A Randomized Controlled Trial of Multicomponent Exercise in Older Adults with Mild Cognitive Impairment

Background

To examine the effect of multicomponent exercise program on memory function in older adults with mild cognitive impairment (MCI), and identify biomarkers associated with improvement of cognitive functions.

Methodology/Principal Findings

Subjects were 100 older adults (mean age, 75 years) with MCI. The subjects were classified to an amnestic MCI group (n = 50) with neuroimaging measures, and other MCI group (n = 50) before the randomization. Subjects in each group were randomized to either a multicomponent exercise or an education control group using a ratio of 1∶1. The exercise group exercised for 90 min/d, 2 d/wk, 40 times for 6 months. The exercise program was conducted under multitask conditions to stimulate attention and memory. The control group attended two education classes. A repeated-measures ANOVA revealed that no group × time interactions on the cognitive tests and brain atrophy in MCI patients. A sub-analysis of amnestic MCI patients for group × time interactions revealed that the exercise group exhibited significantly better Mini-Mental State Examination (p = .04) and logical memory scores (p = .04), and reducing whole brain cortical atrophy (p<.05) compared to the control group. Low total cholesterol levels before the intervention were associated with an improvement of logical memory scores (p<.05), and a higher level of brain-derived neurotrophic factor was significantly related to improved ADAS-cog scores (p<.05).

Conclusions/Significance

The results suggested that an exercise intervention is beneficial for improving logical memory and maintaining general cognitive function and reducing whole brain cortical atrophy in older adults with amnestic MCI. Low total cholesterol and higher brain-derived neurotrophic factor may predict improvement of cognitive functions in older adults with MCI. Further studies are required to determine the positive effects of exercise on cognitive function in older adults with MCI.

Trial Registration

UMIN-CTR UMIN000003662 ctr.cgi&quest;function&hairsp;&equals;&hairsp;brows&amp;action&hairsp;&equals;&hairsp;brows&amp;type&hairsp;&equals;&hairsp;summary&amp;recptno&hairsp;&equals;&hairsp;R000004436&amp;language&hairsp;&equals;&hairsp;J.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. II. Fraction of RNA polymerase engaged in the synthesis of stable RNA at different steady-state growth rates

The relative rates of stable RNA synthesis (rate of stable synthesis/rate of total RNA synthesis) were determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) media. The relative rates were 0.29, 0.50 and 0.66 at these growth rates. From the relative rates, the fraction of RNA polymerase engaged in the synthesis of stable RNA, ψ_s, was calculated to be 0.22, 0.36 and 0.48, respectively, by taking into account the difference between the RNA chain growth rate of stable and that of unstable RNA. The relationship between these ψ_s values and μ and our previously determined chain growth rate of stable RNA has two implications for the control of RNA synthesis during a nutritional shift-up: (1) the increase in the net rate of RNA synthesis after a shift-up results from a transfer of RNA polymerase molecules from unstable to stable RNA genes, and a concomitant increase in the stable RNA chain growth rate, but does not require an activation of RNA polymerase; (2) the synthesis of functioning RNA polymerase enzymes is subject to a growth rate-dependent control.     

Initiation and Velocity of Chromosome Replication in Escherichia coli B/r and K-12

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common Escherichia coli strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (C period) were determined. For these two strains, the initiation mass approached values of 8 × 10⁻¹⁰ and 10 × 10⁻¹⁰ units of optical density (at 460 nm) of culture mass per oriC, respectively, at growth rates above 1 doubling/h (at 37°C). The amount of protein per oriC decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The C period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and C period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.     

Effect of tryptophan on growth and morphology of Hansenula schneggii cells

Sundhagul, Malee (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Effect of tryptophan on growth and morphology of Hansenula schneggii cells. J. Bacteriol. 92:241-249. 1966.-When Hansenula schneggii cells were cultured aerobically in a tryptophan-glucose medium, 70 to 90% of the cells were elongated; no growth occurred under anaerobic conditions. The size of the elongated cells was 15 to 25 mu by 2 to 4 mu, as compared with 2.5 to 5 mu for ellipsoidal cells. Formation of elongated cells occurred essentially during the logarithmic growth period; the highest percentage of elongated cells was found soon after the end of this growth phase. In the later stationary phase, some of the cells formed spherical buds which became spherical cells. The rate of cell division during this period was greatly reduced, but the spherical cells formed decreased the percentage of elongated cells in the population. Cells cultured in a membrane-filter filtrate of a tryptophan-glucose medium (with limiting tryptophan), in which elongated cells had been grown, were ellipsoidal until nitrogenous nutrients were exhausted; thereafter the cells were elongated if tryptophan was added. Of compounds related to tryptophan, kynurenine was the only one which induced a high percentage of the cells to elongate. Some amino acids, such as cystine, histidine, phenylalanine, tyrosine, and threonine, induced elongation in about 15% of the cells. Growth of cells with other amino acids, or the addition of most of the other amino acids to tryptophan-glucose medium, resulted in a population of spherical cells. Several consecutive sequential transfers of cells into tryptophan medium induced elongation in 90% of the cells, but one transfer from a culture with elongated cells into a medium with ammonium sulfate, or a mixture of amino acids, gave a culture with ellipsoidal cells. Growth in media at pH 4 or 5 favored formation of elongated cells; as the pH was increased, the percentage of elongated cells decreased. Carbon sources other than glucose did not affect the percentage of elongated cells, except for the alcohols mannitol and erythitol, which gave comparable growth but reduced the percentage of elongated cells from 70 to 50%. Cell wall analyses of the two types of cells indicated that elongated cells have 2.5 times as much mannan as cell walls of ellipsoidal cells. This suggests that tryptophan, kynurenine, and, to a limited extent, some of the other amino acids cause a diversion of polysaccharide biosynthesis to mannan in the elongated cells rather than to glucan as in ellipsoidal cells.     

Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation.

The uptake and metabolism of 35S-labelled sulphur amino acids were compared in periportal (PP) and perivenous (PV) rat hepatocytes, isolated by digitonin/collagenase perfusion, to identify the factors underlying the previously observed [Kera, Penttilä & Lindros, Biochem. J. (1988) 254, 411-417] higher rate of GSH replenishment in PP cells. The buthionine sulphoximine-inhibitable synthesis of GSH was faster in PP than in PV hepatocytes with both cysteine (6.1 versus 5.0 mumol/h per g of cells) and methionine (4.5 versus 3.3 mumol/h per g) as well as with endogenous precursors and L-2-oxo-4-thiazolidinecarboxylate as substrates. However, the uptake of cysteine by PP cells was slower than by PV cells (8.6 versus 10.3 mumol/h per g of cells), whereas methionine was taken up at similar rates. The activity of gamma-glutamylcysteine synthetase (GCS) was slightly higher in digitonin lysates from the PP than from the PV zone. Production of sulphate, the major catabolite of [35S]cysteine sulphur, as well as incorporation of the label into protein occurred at similar rates in PP and PV cells. Taurine, on the other hand, was produced from [35S]cysteine much faster by PV than by PP cells (0.7 versus 0.1 mumol/h per g of cells). Accordingly, the taurine content of PV hepatocytes tended to be higher and to increase faster during incubation with methionine. These results imply that metabolism of taurine is highly zonated within the acinus. They also suggest that both the slightly lower GCS activity and the fast metabolism of cysteine to taurine limit the capacity of PV hepatocytes to synthesize GSH.     

Synthesis of RNA polymerase in Escherichia coli B-r growing at different rates

Polyacrylamide gel electrophoresis of unfractionated sodium dodecyl sulfate lysates of Escherichia coli$\text{B}\text{r}$ has been used to investigate the synthesis of β and β′ subunits of RNA polymerase as a function of bacterial growth rate. In succinate (μ = 0.67 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) supplemented media, the fraction of [¹⁴C]leucine-labeled β and β′ protein/total protein was found to be 1.05, 1.31 and 1.56%, respectively. Comparison of these values with recent estimates from this laboratory of the differential rate of synthesis of functioning RNA polymerase suggests an excess of total over functioning RNA polymerase. The significance of these data in reference to the regulation of RNA polymerase synthesis is discussed.     

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.     

Polypeptide linkages and resulting structural features as powerful chromogenic factors in the Lowry phenol reaction. Studies on a glycoprotein containing no Lowry phenol-reactive amino acids and on its desialylated and deglycosylated products.

With bovine serum albumin as the reference standard, the armadillo salivary-gland glycoprotein, although containing no chromogenic amino acids and only small amounts of colour-yielding peptides [Chou & Goldstein (1960) Biochem. J. 75, 100-115], is highly reactive in the Lowry phenol protein assay [Wu & Pigman (1977) Biochem. J. 161, 37-47]. After desialylation and Smith degradation of the glycoprotein, the Lowry phenol value increased by 13 and 30% respectively, which suggests that both sialic acid and N-acetylhexosamine exert shielding effects in this reaction. Acid hydrolysis for 30 min decreased the Lowry phenol value by more than 45%, which indicates that the peptide linkages and steric features affect the Lowry phenol reactivity. After hydrolysis for up to 6h, the remaining Lowry phenol value of the partially hydrolysed core protein paralleled the amount of unhydrolysed peptides, inferring that both acid-sensitive and acid-resistant chromophoric peptides are fairly evenly distributed along the whole polypeptide chain. As with bovine serum albumin, more than 80% of the colour yield obtained in the Lowry phenol assay with this glycoprotein is Cu2+-dependent.     

Translation elongation rate varies among organs and decreases with age

There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.     

Amino acid depletion modulates vascular endothelial growth factor production during the life span of human vascular smooth muscle cells

The role of nutrient supply in the replicative capacity and secretory phenotype of cultured human diploid cells is unclear. We examined the relationship between amino acid privation, the secretion of vascular endothelial growth factor (VEGF) and growth phenotype of vascular smooth muscle cells (VSMC), and endothelial cells. Cultures of VSMCs, but not endothelial cells, were growth inhibited by exposure to medium that was 75% deficient in leucine, methionine, arginine, and cysteine over two passages. Exposed VSMC cultures exhibited an increased vulnerability to apoptosis. The maximal cumulative population doubling of the exposed cells was reduced significantly compared with the control cells (25.7 ± 2.0 doublings vs. 27.9 ± 2.1 doublings; P < 0.03). Constitutive VEGF production first became evident in the later passages of the exposed and nonexposed cell cultures. However, production of VEGF was 17-fold greater in the exposed cultures at the tenth passage (P < 0.001). The replicative capacity and constitutive production of VEGF in VSMCs in culture may be programmed by transient privation of amino acids. These observations are relevant to new concepts concerning the pathogenesis of vascular disease. J. Cell. Physiol. 176:359–364, 1998. © 1998 Wiley-Liss, Inc.     

Effects of hypophysectomy, growth hormone, and thyroxine on protein turnover in heart.

Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in vitro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids, glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation. Cathepsin D activity in heart homogenates (+ Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.     

Morphometric evaluation of the specific growth rate of Aspergillus niger grown in agar plates at high glucose levels

Development of surface grown cultures of Aspergillus niger no. 10 was studied at two experimental levels: (a) following the time course of the biomass density (X [=] mg cm(-2)) and fitting the data by the logistic expression, which yielded a macroscopic specific growth rate expressed as mu(obs) = (dX/Xdt)[1-(X/X(max))](-1); and (b) measuring morphometric parameters like the specific elongation rate (k) of the germ tubes and their diameters (D(h)), the colony rate of radial extension (u(r)), and the mean length of distal hyphae (L(av)) to estimate the specific growth rate with the following proposed expression: mu(calc) = u(r)ln2[L(av)ln(L(av)/D(h))](-1). Increases in the initial glucose concentration (10, 40, 70, 120, 200, and 300 g L(-1)) caused reductions in the specific growth rates, the elongation kinetics of the germ tubes, and the hyphal diameter, nevertheless, u(r) and X(max) presented parabolic behavior, showing their maxima in the interval of 90 to 120 g L(-1) of glucose. The overall macroscopic effect of the tested concentrations of glucose on surface grown cultures of A. niger was to produce densely packed and slowly extending colonies, where changes in hyphal lengths and diameters were significant. There was good agreement between mu(obs) and mu(calc) values. Hence, this work validates a kinetic model based on morphometric data to estimate the specific growth rate of molds, obtained from dry weight data, using mold cultures grown in the same solid medium i.e., agar plates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 287-294, 1997.