超高压处理对微生物生理特性的影响

单核增生李斯特菌(Listeriamonocytogenes)NCTC11994和大肠杆菌(Escherichiacoli)ATCC80739经高压处理,其生理特性发生了深刻的变化,主要表现是400MPa以上的压力处理10min,微生物数量下降7个对数单位,压力处理还会导致细胞内pH值的变化,使膜电位下降,细胞内钾流失,ATP浓度降低。     

Systematic analysis of HSP gene expression and effects on cell growth and survival at high hydrostatic pressure in Saccharomyces cerevisiae

We systematically investigated the role of HSP genes in the growth and survival of Saccharomyces cerevisiae under high hydrostatic pressure together with analysis of pressure-regulated gene expression. Cells of strain BY4742 were capable of growth at moderate pressure of 25 MPa. When pressure of 25 MPa was applied to the cells, the expression of HSP78, HSP104, and HSP10 was upregulated by about 3- to 4-fold, and that of HSP32, HSP42, and HSP82 was upregulated by about 2- to 2.6-fold. However, the loss of one of the six genes did not markedly affect growth at 25 MPa, while the loss of HSP31 impaired high-pressure growth. These results suggest that Hsp31 plays a role in high-pressure growth but that the six upregulated genes do not. Extremely high pressure of 125 MPa decreased the viability of the wild-type cells to 1% of the control level. Notably, the loss of HSP genes other than HSP31 enhanced the survival rate by about fivefold at 125 MPa, suggesting that the cellular defensive system against high pressure could be strengthened upon the loss of the HSP genes. In this paper, we describe the requirement for and significance of a subset of HSP genes in yeast cell growth at moderate pressure and survival at extremely high pressure.     

Inhibition of metabolism by hydrostatic pressure: what limits microbial growth?

Summary Analysis of recent and older published data of many investigators indicates that in prokaryotic organisms normally found in environments at atmospheric pressure, the major biological process which is most sensitive to increased hydrostatic pressure is protein synthesis and its inhibition approximately parallels that for growth under the same physical conditions.     

Exploiting the effects of high hydrostatic pressure in biotechnological applications

Applying hydrostatic pressure to biological systems and processes can alter their characteristics. In addition to its use as a basic research tool for investigating the kinetics and thermodynamics of biological systems at the molecular level, the application of pressure is also being used to modify the properties of biological materials to preserve or improve their qualities. This article reviews the principles underlying the observed effects of applied pressure on biological systems, and discusses current and potential application of pressure in biotechnological processes.     

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients

Background

High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates.

Methodology

Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium.Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy.

Principal Findings

A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms.High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.     

Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium

The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured.     

Effect of high hydrostatic pressure on the porin OmpC from Escherichia coli

Porin OmpC from Escherichia coli was reconstituted in liposomes and its gating kinetics were recorded at high hydrostatic pressure, up to 90 MPa, using a development of the patch clamp technique. The composition of the recording solution influenced the results but generally high hydrostatic pressure favoured channel opening.     

The effects of high hydrostatic pressure treatment on the flavor and color of grated ginger

High hydrostatic pressure (HHP) was applied to grated ginger in order to inactivate quality-degrading enzymes in a non-thermal manner. The effects of HHP treatment on the flavor and the color of the grated ginger were investigated just after treatment and during storage. After HHP treatment (400 MPa, 5 min), geraniol dehydrogenase (GeDH) was inactivated to less than 5%, but the activity of polyphenol oxidase (PPO) was reduced only to 37%. Heat treatment (100 °C, 10 min) inactivated GeDH to 43% and PPO to about 10%. In storage, the reduction of geranial, neral, and citronellal to the corresponding alcohols was observed in the untreated and the heat-treated ginger, while it was not in the HHP-treated grated ginger. In the HHP-treated sample, terpene aldehydes almost disappeared without the formation of the corresponding alcohols. Browning was not observed immediately after HHP treatment, while it was complete in the heat-treated sample. The color change during storage appeared to reflect the residual activity of PPO.     

High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Modification of beta-lactoglobulin by microbial transglutaminase under high hydrostatic pressure: localization of reactive glutamine residues

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.     

Cessation of cytokinesis in Schizosaccharomyces pombe during growth after release from high hydrostatic pressure treatment

On the basis of our previous study concerning the effect of high hydrostatic pressure treatment (HPT) on Escherichia coli FtsZ ring (bacterial cytoskeleton) formation, we aimed to determine the effect of HPT on the growth properties of a representative eukaryotic microbe, Schizosaccharomyces pombe, in relation to the behavior of genuine cytoskeletons. Microtubules were visualized with GFP-linked alpha-tubulin. Actin-related cytoskeletons were fluorescently stained with rhodamine-phalloidin. We observed growth retardation of about 10 h in post growth after HPT (75 MPa, 30 min, 28 degrees C), which caused only a little loss of viable cells. In accordance with the period of growth retardation, cessation of cytokinesis and disappearance of the contractile ring (composed of actin, myosin II, and other proteins), directly participates in cytokinesis, continued for 18 h after HPT. On the other hand, the microtubules disappeared only for 6 h after HPT. Based on these observations, the contractile ring was the site most sensitive to HPT resulting in the cessation of cytokinesis.     

Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cells

In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.     

Life under pressure: hydrostatic pressure in cell growth and function

H(2)O is one of the most essential molecules for cellular life. Cell volume, osmolality and hydrostatic pressure are tightly controlled by multiple signaling cascades and they drive crucial cellular functions ranging from exocytosis and growth to apoptosis. Ion fluxes and cell shape restructuring induce asymmetries in osmotic potential across the plasma membrane and lead to localized hydrodynamic flow. Cells have evolved fascinating strategies to harness the potential of hydrodynamic flow to perform crucial functions. Plants exploit hydrodynamics to drive processes including gas exchange, leaf positioning, nutrient acquisition and growth. This paradigm is extended by recent work that reveals an important role for hydrodynamics in pollen tube growth.     

Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology

A comparison of Akt- and Bcl-x(L)-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic gene Bcl-x(L). Although both genes prevent cell death, Akt-protected cells can be distinguished from Bcl-x(L)-protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-x(L)-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-x(L) suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-x(L) maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells.