Changes In Intracellular Composition Research Articles

HIF-1α inhibition by diethylstilbestrol and its polyacetal conjugate in hypoxic prostate tumour cells: insights from NMR metabolomics

In this study, we have employed 1H NMR metabolomics to assess the metabolic responses of PC3 prostate tumour cells to hypoxia and to pharmacological HIF-1α inhibition by DES or its polyacetal conjugate tert-DES. Oxygen deprivation prompted a number of changes in intracellular composition and metabolic activity, mainly reflecting upregulated glycolysis, amino acid catabolism and other compensatory mechanisms used by hypoxic cells to deal with oxidative imbalance and energy deficit. Cell treatment with a non-cytotoxic concentration of DES, under hypoxia, triggered significant changes in 17 metabolites. Among these, lactate, phosphocreatine and reduced glutathione, whose levels showed opposite variations in hypoxic and drug-treated cells, emerged as possible markers of DES-induced HIF-1α inhibition. Furthermore, the free drug had a much higher impact on the cellular metabolome than tert-DES, particularly concerning polyamine and pyrimidine biosynthetic pathways, known to be tightly involved in cell proliferation and growth. This is likely due to the different cell pharmacokinetics observed between free and conjugated DES. Overall, this study has revealed a number of unanticipated metabolic changes that inform on DES and tert-DES direct cellular effects, providing further insight into their mode of action at the biochemical level.

High ethanol production under optimal aeration conditions and yeast composition in a very high gravity fermentation from sweet sorghum juice by Saccharomyces cerevisiae

Aeration is one of the significant factors affecting yeast physiology and impacts ethanol production. The aim of this study was to optimize the aeration rate and time for high levels of ethanol production under very high gravity (VHG) fermentation by Saccharomyces cerevisiae NP 01 using a factorial design based on response surface methodology (RSM). The changes in intracellular composition (trehalose and ergosterol) during the fermentation were also investigated. The ethanol production medium was sweet sorghum juice containing 280gl−1 of total sugar and 3.45gl−1 of urea. The fermentation was carried out at 30°C in a 2‐L fermenter. The results showed that the optimal aeration rate and aeration time for the VHG fermentation were 0.31vvm and 12.01h, respectively. Under the optimal conditions, the ethanol concentration (PE), productivity (Qp) and yield (Yp/s) were 127.80gl−1 (16.20%, v/v), 2.66gl−1h−1 and 0.49gg −1, respectively, whereas the PE values were 119.64 and 79.74gl−1 under no aeration with urea (positive control) and without urea (negative control) supplementation, respectively. The trehalose contents under optimal, positive and negative control conditions were 200, 192 and 154mgg DCW (dry cell weight)−1, respectively. The ergosterol contents under the optimal conditions were 3.35 and 5.74 fold higher than those of positive and negative controls, respectively. It can be conclude that ethanol fermentation efficiency is markedly related to the degree of aeration, trehalose accumulation and ergosterol synthesis in the plasma membranes of yeast cells.

NTiO2 induced changes in intracellular composition and nutrient stoichiometry in primary producer — cyanobacteria

The widely and increasing use of nano-titanium dioxide (nTiO2) has led to its release in the environment and concerns of consequent impact on aquatic eco-relevant biota. Previous studies indicated possible physiological changes (i.e., nitrogen storage) induced by nano-titanium dioxide (nTiO2) exposure in algae, which will likely have ecological implications. This study investigated the short- (96h) and long-term (21days) ecotoxic impact of environmentally relevant nTiO2 concentrations on the cellular biochemical pools and nutrient stoichiometry in the nitrogen-fixing cyanobacteria Anabaena variabilis. Changes in nutrient element ratios and cellular composition were analyzed using both chemical elemental analysis and Fourier Transform Infrared (FT-IR) spectroscopy. Chemical elemental analysis showed that exposure to nTiO2 at varying dose concentrations and exposure duration led to statistically significant changes in intracellular C:N, C:P and N:P stoichiometries compared with those in the controls. In general, there seemed to be a decreasing trends of cellular C:N ratio and increase in the cellular C:P and N:P ratios with the increasing level of nTiO2 exposure. Further FT-IR analysis results revealed both temporal and dose-dependent change patterns of major macromolecules, including protein, lipids, nucleic acids and carbohydrates, in A. variabilis upon nTiO2 exposure. The relative ratio of amide II, lipids, nucleic acids and carbohydrates to the cellular protein content (quantified as amide I stretch) changed significantly within the initial 96h of exposure and, both the magnitude of changes and levels of recovery seemed to be nTiO2 dose-dependent. This study, for the first time, demonstrated that the intracellular composition and nutrient stoichiometry changes could be induced by long-term and short-term exposures to nTiO2 to primary producers, which may have ecological implications for interspecies equilibriums and community dynamics in aquatic ecosystems.

Immunomodulatory synthetic dipeptide L-Glu-L-Trp slows down aging and inhibits spontaneous carcinogenesis in rats.

Immunomodulatory molecule L-Glu-L-Trp was isolated from natural calf thymic peptide complex Thymalin by reverse-phase high performance liquid chromatography. On the basis of the synthesized dipeptide a pharmaceutical was designed containing this compound, which later receives the brand name Thymogen. The agent activated T-cell differentiation, T-cell recognition of peptide-MHC complexes, induced changes in intracellular composition of cyclic nucleotides, and activated neutrophilic chemotaxis and phagocytosis. The effect of dipeptide on survival, life span and spontaneous tumor development was studied in female rats. Seventy-six, five-month-old outbred female rats were randomly subdivided into two groups and were subcutaneously injected with 0.2 ml of normal saline (controls, 32 rats) or with 5 micrograms/rat of the dipeptide L-Glu-L-Trp, dissolved in 0.2 ml of saline (44 rats), 5 times per week for 12 months. Animals were monitored up to their natural death and all the tumors discovered were studied microscopically. Mean life span of rats in both groups was similar but that of 10% maximum survived control rats constituted 949 +/- 16.1 days, whereas in the dipeptide-treated rats this value was 1048 +/- 21.1 days (P < 0.001). Six out of 44 rats treated with the drug survived over the maximum life span of control rats (965 days). The aging rate indicated as alpha in the Gompertz equation, was 0.0071 days-1 in controls and 0.0041 days-1 in rats exposed to L-Glu-L-Trp. Total tumor incidence was 1.5 times lower (P < 0.01), malignant tumor incidence 1.7 times lower (P < 0.01), and hematopoietic malignancies (leukemias and lymphomas) 3.4 times lower (P < 0.02) in rats exposed to the dipeptide in comparison with controls. Thus, treatment with L-Glu-L-Trp delayed aging rate and decreased spontaneous tumor incidence in rats.

Progressive changes in cortical water and electrolyte content at three stages of rat infantile hydrocephalus and the effect of shunt treatment.

Infantile hydrocephalus causes injury to the developing brain and despite surgical treatment, neurological deficits persist. The H-Tx rat develops inherited hydrocephalus in late gestation. Rapid postnatal ventricular enlargement, results in severe hydrocephalus by 21 days after birth. This is accompanied by changes in cortical morphology and metabolite content that indicate possible changes in intracellular composition. This study has tested the hypothesis that tissue water and electrolyte content is altered in hydrocephalus. The objective was to gain further insight into the mechanisms leading to neuronal damage. Water and electrolyte content (Na+, Cl-, and K+) were measured in the cerebral cortex of control and hydrocephalic rats at 4, 11, and 21 days after birth, and at 21 days in rats that received alleviating shunt surgery at 4 or 11 days. At all ages, hydrocephalic tissue was significantly increased over control for cortical water, Na+, and Cl- content. Additionally, at the intermediate (11-day) and advanced (21-day) stages there were significant decreases in K+ content, consistent with previous observations of decreases in organic osmolytes and energy metabolites. This suggests that by 11 days there are intracellular changes, probably through impaired membrane homeostatic mechanisms. In shunt-treated rats, the extracellular constituents were almost normal, although a small increase over control values persisted. The decrease in intracellular K+ was not corrected in either group of shunt-treated rats. It is concluded that early hydrocephalus is characterized by extracellular edema that largely reverses with shunt treatment. Subsequently, as the hydrocephalus progresses, there is a breakdown of cell homeostasis and an irreversible loss of intracellular constituents.

Natural and synthetic thymic peptides as therapeutics for immune dysfunction

Natural thymic peptides have been isolated from calf thymus by mild acid extraction. Pharmaceutical containing natural peptides (Thymalin ®) was put into practice as immunocorrector. One of the immunomodulatory molecules ( l-Glu- l-Trp) has been isolated from Thymalin by reversed-phase high performance liquid chromatography. Pharmaceutical containing this agent (Thymogen ®) was designed on the base of synthesized dipeptide. A novel immunomodulatory dipeptide was synthesized and termed Vilon ®. Both natural and synthetic pharmaceuticals activated T-cell differentiation, T-cell recognition of peptide-MHC complexes, induced the changes in intracellular composition of cyclic nucleotides and cytokine [interleukin (IL-2), interferon (IFN)] excretion of blood lymphocytes. Synthetic dipeptides activated neutrophil chemotaxis and phagocytosis. They had no influence on antioxidant response in thymocytes in comparison with natural peptides. Thymalin and Thymogen were used in persons with chronic pathology and immune dysfunction. The results indicate that thymic peptides participate in the regulating mechanisms of inflammatory processes as cytokine antagonists and show the difference between natural and synthetic products. It is important for the drugs designed to prevent immune dysfunction development.

MICROSCALE HEAT TRANSFER IN BIOLOGICAL SYSTEMS AT LOW TEMPERATURES

This review of heat and mass transfer in biological systems is focused on phenomena occurring at low temperatures at the micro- and nano-scates. The discussed temperature range is divided into temperatures above freezing and temperatures below freezing. At temperatures above freezing, the effects of low temperature relate to changes in the molecular structure of cell membranes, changes in ionic pump function, and chemical reaction kinetics. Below freezing, processes relate to water transport across cell membranes and changes in intracellular composition. The mechanisms through which organisms survive changes induced by low temperatures are also discussed together with biomedical applications of low temperatures in cryopreservation of organs for transplantation and destruction of undesirable tissues by freezing in cryosurgery.

Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Changes in intracellular composition after hyperosmotic shock were studied in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032. Both strains accumulated betaine, proline, glutamic acid, glutamine and trehalose in response to stress. The accumulated amino acids were synthesized by the cells, while betaine and trehalose were taken up from the medium. The contribution of synthesized osmoregulators was highest in C. glutamicum. In a sucrose-limited continuous culture, the increased outer osmotic pressure was balanced within 15 min for C. glutamicum and somewhat later in B. lactofermentum. The rapid regulation was due to both accumulation of osmoregulators, and shrinkage of cell and cytoplasmic volume. Immediately after shock, glutamine and glutamic acid were the dominating osmolytes. During the adaptation process, glutamine was replaced by the better osmoprotectant proline. In betaine-enriched cultures, betaine accumulation increased at the expense of glutamic acid, glutamine and trehalose. The total intracellular concentration of osmolytes increased linearly with increasing stress for all stress factors.

Acute changes in intracellular ions or pH and regulation of aldose reductase activity.

Sorbitol production in the renal medulla increases in dehydrated rats, indicating that aldose reductase activity varies with the state of hydration. This response could be due to an increased synthesis of the enzyme (Moriyama T et al. J Biol Chem 1989:264:16810-16814) and/or a change in aldose reductase activity caused by acute changes in intracellular ionic composition, ionic strength, osmolality, or pH. Aldose reductase activity in tubules dissected from kidneys of control rats and rats undergoing water diuresis was measured, and the tubules were permeabilized so that changes in intracellular composition that would occur during dehydration could be induced experimentally. Aldose reductase activity did not change consistently as sodium, potassium, chloride, or osmolality were varied. Aldose reductase activity did increase acutely when sulfate was raised or when pH was lowered to pH 6.2 to 6.8, corresponding to the pH optimum of the enzyme. The small magnitude of change in enzyme activity suggests that the major influence of dehydration on aldose reductase activity is to increase enzyme synthesis. It was concluded that aldose reductase activity is not acutely regulated by changes in sodium, potassium, chloride, or osmolality. The stability of aldose reductase activity despite changes in ionic composition or osmolality supports the hypothesis that acute regulation of intracellular sorbitol content occurs by variation in cell sorbitol permeability and not by variation in cell sorbitol production.

In Vivo NMR Analysis of the Influence of Pyruvate Decarboxylase and Alcohol Dehydrogenase of Zymomonas mobilis on the Anaerobic Metabolism of Escherichia coli

AbstractPhosphorus‐31 NMR studies of Escherichia coli expressing cloned pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes from Zymomonas mobilis revealed significant changes in the concentration of intracellular metabolites. The intracellular concentrations of phosphate, sugar phosphates, NAD(H), NTP, NDP, and UDPG of the strain HB101 expressing pdc, adh, or both genes were analyzed at quasi steady state during the anaerobic catabolism of glucose. The evolution of the intracellular pH and extracellular pH during glucose consumption showed a constant value of ΔpH equal to 0.2 pH unit that did not depend on the expression of the Zymomonas genes. The strains expressing PDC only exhibited greatest changes in intracellular composition with significantly decreased inorganic phosphate, NAD(H), NTP, and UDPG and significantly increased sugar phosphates. Analysis of the fermentation products during the NMR experiments provided further information about the physilogical changes observed in the cells. Two plasmid effects on the host metabolism are reported, one induced by the plasmid utilized as control (pUCl9), and the other induced by the expression of the pet operon. In both cases an enhancement of the glucose uptake rate was observed. Although the plasmid carrying adh induces a rapid metabolic inhibition, the expression of pdc does not. The expression of pdc and adh together showed a physiological enhancement that is attributable not to effects on external pH but rather to the effect of the switch in pathways from mixed acid to ethanol fermentation.

Use of Human Minor Salivary Glands in Basic and Applied Secretion Research

Previous findings from studies utilizing human labial and palatine minor salivary glands are reviewed. These studies took histopathological, biochemical, and ultrastructural approaches, and focused on control and diseased glands. Disease-oriented summarizations are used, and control results are discussed in the context of disease-related findings. Findings are reviewed separately for electrolytes, macromolecules, and ultrastructure. In control subjects, minor gland salivary electrolyte concentrations are dependent on flow rate, and this dependence may be altered by diseases such as cystic fibrosis as-well as by inflammatory situations such as graft-versus-host disease. There is also evidence that salivary electrolyte secretion processes are not similar in labial and palatine minor glands. Studies of salivary macromolecular composition are reviewed for control subjects and for patients with graft-versus-host disease and Sjögren's syndrome. The findings indicate that the macromolecular contents of labial and palatine gland saliva are similar, but that both are significantly different from that for major gland saliva. Finally, studies attempting to measure disease-related changes in intracellular composition are reviewed. It is concluded that the minor salivary glands are important models for the study of exocrine gland physiology and pathophysiology in man.

Use of human minor salivary glands in basic and applied secretion research.

Previous findings from studies utilizing human labial and palatine minor salivary glands are reviewed. These studies took histopathological, biochemical, and ultrastructural approaches, and focused on control and diseased glands. Disease-oriented summarization are used, and control results are discussed in the context of disease-related findings. Findings are reviewed separately for electrolytes, macromolecules, and ultrastructure. In control subjects, minor gland salivary electrolyte concentrations are dependent on flow rate, and this dependence may be altered by diseases such as cystic fibrosis as well as by inflammatory situations such as graft-versus-host disease. There is also evidence that salivary electrolyte secretion processes are not similar in labial and palatine minor glands. Studies of salivary macromolecular composition are reviewed for control subjects and for patients with graft-versus-host disease and Sjögren's syndrome. The findings indicate that the macromolecular contents of labial and palatine gland saliva are similar, but that both are significantly different from that for major gland saliva. Finally, studies attempting to measure disease-related changes in intracellular composition are reviewed. It is concluded that the minor salivary glands are important models for the study of exocrine gland physiology and pathophysiology in man.

Cell size as an indicator of changes in intracellular composition of Azotobacter vinelandii.

Cell size, measured electronically, was correlated to changes in cellular composition, number, and morphology of Azotobacter vinelandii OP during batch growth. The effect of a changing abiotic environment on these features of the cell is discussed. For this organism exponential growth was unbalanced growth and cell-size change was a sensitive indicator of this growth pattern. Cell-size measurements have the potential to give a rapid assessment of intracellular compositional changes.