scholarly journals Exact distribution of threshold-crossing times for protein concentrations: Implication for biological timekeeping

2021 ◽  
Author(s):  
Krishna Rijal ◽  
Ashok Prasad ◽  
Abhyudai Singh ◽  
Dibyendu Das
Keyword(s):  
Protein Concentration ◽  
Exact Distribution ◽  
Protein Accumulation ◽  
Good Match ◽  
E Coli ◽  
Physiological Processes ◽  
Lysis Time ◽  
Threshold Crossing ◽  
Concentration Threshold ◽  
Mean Variance

Stochastic transcription and translation dynamics of protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold-crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance and skewness. The distribution is asymmetric and its skewness non-monotonically varies with the threshold. We study lysis times of E-coli cells for holin gene mutants of bacteriophage-λ and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted.

scholarly journals Anti-EGFR VHH Antibody under Thermal Stress is Better Solubilized with a Lysine than with an Arginine SEP Tag

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 810
Author(s):  
Md. Golam Kibria ◽  
Akari Fukutani ◽  
Yoko Akazawa-Ogawa ◽  
Yoshihisa Hagihara ◽  
Yutaka Kuroda
Keyword(s):  
Thermal Stress ◽  
Phosphate Buffer ◽  
Protein Concentration ◽  
Biochemical Analysis ◽  
Stress Condition ◽  
Binding Activity ◽  
Denatured State ◽  
E Coli ◽  
Structural And Functional Properties ◽  
Model Protein

In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

DNA methylation in Yersinia enterocolitica: role of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2291-2299 ◽  
Author(s):  
Stefan Fälker ◽  
M. Alexander Schmidt ◽  
Gerhard Heusipp
Keyword(s):  
Mismatch Repair ◽  
Yersinia Enterocolitica ◽  
Virulence Genes ◽  
Spontaneous Mutation ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Physiological Processes ◽  
Dna Adenine Methyltransferase

DNA adenine methyltransferase (Dam) plays an important role in physiological processes of Gram-negative bacteria such as mismatch repair and replication. In addition, Dam regulates the expression of virulence genes in various species. The authors cloned the dam gene of Yersinia enterocolitica and showed that Dam is essential for viability. Dam overproduction in Y. enterocolitica resulted in an increased frequency of spontaneous mutation and decreased resistance to 2-aminopurine; however, these effects were only marginal compared to the effect of overproduction of Escherichia coli-derived Dam in Y. enterocolitica, implying different roles or activities of Dam in mismatch repair of the two species. These differences in Dam function are not the cause for the essentiality of Dam in Y. enterocolitica, as Dam of E. coli can complement a dam defect in Y. enterocolitica. Instead, Dam seems to interfere with expression of essential genes. Furthermore, Dam mediates virulence of Y. enterocolitica. Dam overproduction results in increased tissue culture invasion of Y. enterocolitica, while the expression of specifically in vivo-expressed genes is not altered.

Forage productivity of smooth brome grass mixtures under the application of growth regulators

2021 ◽  
pp. 9-15
Author(s):  
В.Г. Васин ◽  
М.С. Кригер ◽  
С.А. Васин
Keyword(s):  
Growth Regulators ◽  
Crop Production ◽  
Protein Concentration ◽  
Matter Content ◽  
Energy Yield ◽  
Protein Accumulation ◽  
Dry Matter Content ◽  
Smooth Brome ◽  
Arable Farming ◽  
Brome Grass

Исследования проведены в 2019–2020 годах в экспериментальном севообороте научно-исследовательской лаборатории «Корма» кафедры «Растениеводство и земледелие» Самарского ГАУ. В статье представлены данные по кормовой продуктивности травосмесей костреца безостого с кострецом прямым, бобовыми травами и черноголовником многобрачным при применении стимуляторов «Матрица роста» и «Гуми-20М». В состав исследуемых травосмесей входили эспарцет песчаный, люцерна посевная и лядвенец рогатый. Посев проведён в мае 2015 года. Исследование охватывало фазу вымётывания костреца безостого и прямого и цветения бобовых, во время которой оценивалась урожайность, определялся химический состав травостоев (особое внимание уделялось содержанию протеина и динамике его изменения), а также проводился учёт кормовых достоинств (накопления сухого вещества, переваримого протеина и выхода обменной энергии). Высокую продуктивность формировали трёх- и четырёхкомпонентные травостои с эспарцетом песчаным и люцерной посевной. Травостои с лядвенцем рогатым обеспечивали максимальные показатели. Наблюдалось отчётливое увеличение всех изучаемых показателей при добавлении бобового компонента. Продуктивность обработанных стимуляторами травосмесей была, как правило, больше, чем в контрольном варианте. Минимальную продуктивность формировали смеси, в которых присутствовали только злаковые компоненты и которые не обрабатывались стимуляторами роста. Также выявлено, что содержание протеина в травостоях с бобовым компонентом было выше, чем в травостоях с мятликовыми культурами, обработка посевов стимуляторами способствовала повышению протеина. Анализ доли компонентов в травостоях показал, что злаковый компонент преобладал над бобовым, однако в некоторых вариантах наблюдалось преобладание эспарцета и люцерны над злаками. Наименьшую долю в травостое составляли лядвенец рогатый и черноголовник многобрачный. Зависимости процентного соотношения компонентов от варианта обработки выявлено не было. The investigation took place at the laboratory “Korma“ of the Samara State Agrarian University (the department of Crop Production and Arable farming) in 2019–2020. The article reports on the productivity of grass mixtures of smooth brome with erect brome, legumes and fodder burnet under the application of growth regulators “Matritsa rosta“ and “Gumi-20M“. The mixtures also contained hungarian sainfoin, alfalfa and birdʼs-foot trefoil. Crops were planted in May 2015. Crops were tested at the heading or flowering stages. The following traits were analyzed: productivity, chemical composition, dry matter content, crude protein and exchange energy yield. The dynamics of protein accumulation was studied. Three- and four-component grass mixtures with hungarian sainfoin and alfalfa showed the highest productivity. Mixtures with birdʼs-foot trefoil performed the best. Introduction of legumes into swards positively affected all the traits studied. Generally, the application of growth regulators resulted in higher productivity. Gramineous mixtures had the lowest productivity under no treatment. Swards with legumes provided more protein. The treatment with growth regulators increased protein concentration. Gramineous dominated legumes, however, in some variants hungarian sainfoin and alfalfa were predominant. Birdʼs-foot trefoil and fodder burnet had the smallest share in swords. There was no significant correlation between mixture composition and treatments.

Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

2021 ◽  
Author(s):  
Dimitar Plamenov Petrov ◽  
Steffen Kaiser ◽  
Stefanie Kaiser ◽  
Kirsten Jung
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Gel Electrophoresis ◽  
E Coli ◽  
Physiological Processes ◽  
Mrna Methylation ◽  
Important Regulator ◽  
Quantitative Profiling

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

scholarly journals Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vishal Srivastava ◽  
Shivam Mishra ◽  
Tapan K. Chaudhuri
Keyword(s):  
Protein Expression ◽  
Process Parameters ◽  
Recombinant Expression ◽  
Cellular Protein ◽  
Biologically Active ◽  
Fixed Dose ◽  
Antibiofilm Activity ◽  
Protein Accumulation ◽  
E Coli ◽  
Enhanced Production

Abstract Background Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation. Results Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification. Conclusion Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

Comparison of lactational responses of dairy cows in Georgia and Israel to heat load and photoperiod

2002 ◽  
Vol 75 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Y. Aharoni ◽  
O. Ravagnolo ◽  
I. Misztal
Keyword(s):  
Milk Yield ◽  
Heat Load ◽  
Protein Concentration ◽  
Day Length ◽  
Seasonal Effects ◽  
Good Match ◽  
Negative Effects ◽  
Positive Effects ◽  
Daily Change ◽  
Positive Effect

AbstractThe seasonal effects of heat load and photoperiod on yield and composition of milk from primiparous cows in the course of lactation were studied using test day records from 8968 primiparous cows on 76 farms in Georgia, collected from 1990 through 1997. The effect of prepartum photoperiod on milk production in the subsequent lactation of these cows was also evaluated. These estimated seasonal effects were compared with those estimated for 4728 primiparous cows on 13 farms, and for 1538 multiparous cows on three farms during consecutive lactations in Israel from 1994 through 1996. During lactation, the day length had a positive effect on milk yield and negative effects on fat and protein concentrations in the milk, but the daily change in day length had positive effects on milk yield and fat concentration, and a smaller positive effect on protein concentration. The day length during the prepartum period had negative effects on milk yield and fat and protein concentrations. The heat load during lactation had negative effects on milk yield and fat and protein concentrations. Most of the effects were highly (P < 0·001) significant. There was a very good match between the results obtained for primiparous cows in Georgia and Israel, for the combined effects of heat load and photoperiod during lactation on milk yield and protein and fat concentrations. The match between primiparous and multiparous cows in Israel was better for milk yield and protein concentration than for fat concentration. The estimated effects of pre-partum photoperiod were higher for multiparous cows in Israel than for primiparous cows in either country.

Diphenhydramine reduces endotoxin effects on lung vascular permeability in sheep

1980 ◽  
Vol 49 (3) ◽  
pp. 516-520 ◽  
Author(s):  
K. L. Brigham ◽  
S. J. Padove ◽  
D. Bryant ◽  
C. R. McKeen ◽  
R. E. Bowers
Keyword(s):  
Vascular Permeability ◽  
Protein Concentration ◽  
Base Line ◽  
E Coli ◽  
P Protein ◽  
Endogenous Histamine ◽  
Pulmonary Arterial ◽  
Pressure Changes ◽  
Protein Rich ◽  
Lung Vascular Permeability

Because, in sheep, histamine-induced increased lung vascular permeability is prevented by diphenhydramine, we tested the effects of diphenhydramine on the sheep lung vascular response to endotoxin. We infused E. coli endotoxin (0.40-1.00 micrograms/kg) with and without diphenhydramine (3.0 mg/kg bolus + 1.5 mg . kg-1 . h-1) in the same unanesthetized sheep while measuring pulmonary arterial (Ppa) and left atrial (Pla) pressures, lung lymph flow (Qlym) and lymph (L) and plasma (P) protein concentrations. Endotoxin caused pulmonary hypertension soon after infusion (base-line Ppa = 22 +/- 3 (SE) cmH2O; after endotoxin Ppa = 40 +/- 2; P less than 0.05, n = 6) and after several hours an increase in permeability reflected in high flow of protein-rich lymph (base-line; Qlym = 7.5 +/- 1.4 (SE) ml/h, L/P protein concentration = 0.60 +/- 0.02: after endotoxin; Qlym = 21.4 +/- 3.1, P less than 0.05; L/P = 0.66 +/- 0.03, P less than 0.05). In the presence of diphenhydramine, endotoxin caused identical pressure changes but Qlym was lower during the period of increased permeability (16.7 +/- 3.0 (SE) ml/h, P less than 0.05 compared to endotoxin alone) and L/P protein concentration was similar (0.68 +/- 0.04, P = NS). We conclude that endogenous histamine may be partly responsible for the increase in lung vascular permeability after endotoxemia, but that histamine probably is not the sole mediator of the permeability change.

scholarly journals How to Avoid a No-Deal ER Exit

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1051 ◽  
Author(s):  
Tiziana Anelli ◽  
Paola Panina-Bordignon
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Protein Concentration ◽  
Secretory Proteins ◽  
Protein Accumulation ◽  
Secreted Protein ◽  
Native Structure ◽  
Golgi Compartment ◽  
Abnormal Increase ◽  
Er Exit Sites

Efficiency and fidelity of protein secretion are achieved thanks to the presence of different steps, located sequentially in time and space along the secretory compartment, controlling protein folding and maturation. After entering into the endoplasmic reticulum (ER), secretory proteins attain their native structure thanks to specific chaperones and enzymes. Only correctly folded molecules are allowed by quality control (QC) mechanisms to leave the ER and proceed to downstream compartments. Proteins that cannot fold properly are instead retained in the ER to be finally destined to proteasomal degradation. Exiting from the ER requires, in most cases, the use of coated vesicles, departing at the ER exit sites, which will fuse with the Golgi compartment, thus releasing their cargoes. Protein accumulation in the ER can be caused by a too stringent QC or by ineffective transport: these situations could be deleterious for the organism, due to the loss of the secreted protein, and to the cell itself, because of abnormal increase of protein concentration in the ER. In both cases, diseases can arise. In this review, we will describe the pathophysiology of protein folding and transport between the ER and the Golgi compartment.

A fraction from Escherichia coli with anti-Aspergillus properties

2005 ◽  
Vol 54 (4) ◽  
pp. 375-379 ◽  
Author(s):  
V Yadav ◽  
R Mandhan ◽  
Rajesh Dabur ◽  
A K Chhillar ◽  
J Gupta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Exchange Chromatography ◽  
Active Molecule ◽  
Standard Drug ◽  
E Coli ◽  
Variable Activity ◽  
Sds Page ◽  
Haemolytic Assay

The products of various strains of Escherichia coli (BL21, DH5α, HB101 and XL Blue) were investigated for antimycotic properties using pathogenic isolates of Aspergillus. Co-culture experiments revealed that E. coli strains exhibited variable activity against Aspergillus fumigatus. The lysates prepared from DH5α, HB101 and XL Blue strains of E. coli showed inhibitory activity against A. fumigatus in the protein concentration range of 62.50 to 250.00 μg ml−1. The highest activity was seen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completely at a concentration of 31.25 μg protein ml−1. The MIC of BL21 lysate against Aspergillus niger was found to be 62.50 μg ml−1. The in vitro toxicity of BL21 lysate was evaluated using a haemolytic assay. A BL21 lysate protein concentration of 1250.00 μg ml−1 was found to be nontoxic to human erythrocytes. The standard drug amphotericin B lysed 100 % of erythrocytes at a concentration of 37.50 μg ml−1. SDS-PAGE showed the presence of at least 15 major proteins in the lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions and fraction III was found to be endowed with anti-Aspergillus properties. The MIC of this fraction was found to be 3.90 μg ml−1. Further work on the purification of the active molecule and its characterization is in progress.

scholarly journals Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016 ◽  
Vol 198 (12) ◽  
pp. 1735-1742 ◽  
Author(s):  
Nastaran Hadizadeh ◽  
Reid C. Johnson ◽  
John F. Marko
Keyword(s):  
Gene Regulation ◽  
Dna Binding ◽  
Protein Concentration ◽  
Model Systems ◽  
Bacterial Chromosome ◽  
Regulatory Function ◽  
Content Type ◽  
E Coli ◽  
Dna Double Helix

ABSTRACTOff-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the majorEscherichia colinucleoid protein Fis on isolated bacterial chromosomes. We isolatedE. colinucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant (kexch) of ≈104M−1s−1, comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observedin vitroon chromosomes assembledin vivo.IMPORTANCEBacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolatedE. colichromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed inin vitroexperiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

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