Cytological and physiological changes related to cryotolerance in recalcitrant <Emphasis Type="Italic">Livistona chinensis</Emphasis> embryos during seed development

Cytological and biochemical changes in recalcitrant Livistona chinensis embryos following the acquisition and loss of cryotolerance to liquid nitrogen during seed development were studied. The embryonic cells were always hydrated and contained fully functional organelles throughout seed development. However, the central cells in the root-epicotyl end of the embryo exhibited partial dedifferentiation during the middle developmental stages, although extensive reduction of mitochondria and vacuolation and intensive accumulation of starch grains, lipid, and protein bodies were not observed. Total soluble sugar content rose then decreased on a fresh weight and water weight basis, while soluble and heat-stable proteins increased in number and content then decreased, as seeds matured. These cytological and biochemical features differ from those of orthodox seeds, providing a physiological basis for the recalcitrant behavior of L. chinensis seeds. The changes were closely correlated with acquisition and loss of cryotolerance in L. chinensis embryos and are presumed to contribute to cryotolerance, which would account for the cryotolerance variation in L. chinensis embryos. Cryotolerance is suggested to be a complex, multifaceted process, and accumulation of soluble sugars and soluble and heat-stable proteins alone is not enough to increase cryotolerance per se without acting in combination with a decrease of cellular metabolic activity.     

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

Flavonoid transport by mammalian endothelial cells

Despite the ever-growing body of literature reporting the effects of flavonoids on animals at both the cellular and systemic levels, one of the most basic questions-"Are the effects of flavonoids on animal cells initiated through their interaction with extracellular targets or intracellular targets?"-has yet to be addressed. Because many effects of flavonoids on cells can be detected within minutes of flavonoid application and because flavonoids diffuse across lipid membranes slowly or not at all, intracellular mechanisms would necessitate a flavonoid transport system for rapid flavonoid uptake. The specific aims of this investigation were (1) to determine if endothelial cells contain a mechanism that mediates rapid flavonoid uptake and (2) to provide evidence for or against the hypothesis that rapid flavonoid effects on endothelial cell synthesis of prostacyclin and endothelin are initiated through the interaction of flavonoids with intracellular targets. Data show that bovine and human aortic endothelial cells possess a transport system that mediates rapid uptake of the flavonoid morin and suggest that the flavonoid uptake system utilizes a variety of oxygenated phenolic compounds as substrates. Further investigation into flavonoid transport should expedite future investigation into the mechanisms of flavonoid actions, because it may allow research to focus on the cellular locations where flavonoids are concentrated. Although endothelial cells contain a mechanism for the rapid uptake of morin, data reported herein suggest that morin initiates its rapid effects on endothelial cell synthesis of prostacyclin and endothelin through an interaction with extracellular targets.     

Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry

The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract.     

Transport of water from concentrated to dilute solutions in cells of Nitella

The transport of water from concentrated to dilute solutions which occurs in the kidney and in a variety of living cells presents a problem of fundamental importance. If the cell acts as an osmometer we may expect to bring about such transport by creating an inwardly directed osmotic drive which is higher in one part of the cell than in other regions of the same cell. The osmotic drive is defined as the difference between internal and external osmotic pressure. Experiments with Nitella show that this expectation is justified. If water is placed at one end of the cell (A) and 0.4 M sucrose with an osmotic pressure of 11.2 atmospheres at the other end (B) water enters at A, passes along inside the cell, and escapes at B leaving behind at B the solutes which cannot pass out through the protoplasm. Hence the internal osmotic pressure becomes much higher at B than at A. When 0.4 M sucrose at B is replaced by 0.3 M sucrose with an osmotic pressure of 8.1 atmospheres we find that water enters at B, passes along inside the cell, and escapes at A so that water is transported from a concentrated to a dilute solution although the difference in osmotic pressure of the 2 solutions is more than 8 atmospheres. The solution at B thus becomes more concentrated. It is evident that if metabolism produces a higher osmotic pressure and consequently a higher inwardly directed osmotic drive in one region of the cell as compared with other parts of the same cell water may be transferred from a concentrated to a dilute solution so that the former solution becomes still more concentrated.     

Single-file diffusion through K+ channels in frog skin epithelium

Summary The ratio between the unidirectional fluxes of K⁺ across the frog skin with K-permeable outer membranes was determined in the absence of Na⁺ in the apical solutions. The experiments were performed under presteady-state conditions to be able to separate the flux ratio for K⁺ through the cells from contributions to the fluxes through extracellular leaks. The cellular flux ratio deviated strongly from the value calculated from the flux ratio for electrodiffusion. The experiments can be explained if the passive K transport through the epithelial cells proceeds through specific channels by single-file diffusion with a flux ratio exponent of about 2.5.     

Extracellular and Metabolic Factors Affecting the Efflux and Influx of Erythrocyte Water

Water exchanges between rabbit erythrocytes and extracellular solutions equidistant from intracellular osmolarity were studied by freezing point depression techniques. Water efflux was always less than water influx and both were hypotonic to the intracellular and extracellular fluids. The magnitudes of these water exchanges were not dependent on the presence of extracellular cation. Stimulation of oxygen uptake by the addition of glucose and methylene blue increased water influx and, possibly, decreased water efflux. This could not be accounted for by accumulation of osmotically active intracellular metabolic products. O₂ uptake was markedly decreased during cellular dehydration, was slightly decreased during cellular overhydration, and was maximum at the water content of erythrocytes when suspended in a medium isotonic with plasma.     

Paxillin: A cytoskeletal target for tyrosine kinases

Paxillin is a recently identified member of the complex of cytoskeletal proteins that is found concentrated in cultured cells and in vivo at the cytoplasmic face of regions of cell attachment to the extracellular matrix. These sites, in view of their close proximity to the extracellular matrix, are well positioned to act as signal-transducing centers to ‘report on’ changes in the cells, immediate environment. Recent findings indicate that such signals are in part mediated through the activation of tyrosine kinases concentrated at the sites of adhesion. Changes in the phosphotyrosine content of paxillin accompanying this elevation in kinase activity suggest that paxillin may be an important intermediary in these pathways.     

Exosome platform for diagnosis and monitoring of traumatic brain injury

We have previously demonstrated the release of membranous structures by cells into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. With activation, injury, stress, transformation or infection, cells express proteins and RNAs associated with the cellular responses to these events. The exosomes released by these cells can exhibit an array of proteins, lipids and nucleic acids linked to these physiologic events. This review focuses on exosomes associated with traumatic brain injury, which may be both diagnostic and a causative factor in the progression of the injury. Based on current data, exosomes play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with development, progression and therapeutic failures and cellular stress in a variety of pathologic conditions. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodelling, signal pathway activation through growth factor/receptor transfer, chemoresistance, immunologic activation and genetic exchange. These circulating exosomes not only represent a central mediator of the pro-inflammatory microenvironment linked with secondary brain injury, but their presence in the peripheral circulation may serve as a surrogate for biopsies, enabling real-time diagnosis and monitoring of neurodegenerative progression.     

Hydrodynamics of concentrated proteoglycan solutions

The dynamics of water transport in proteoglycan compartments has been studied in relation to osmotic flow (proteoglycan diffusion) and hydraulic permeability (proteoglycan sedimentation) in concentrated solutions of proteoglycan subunit and native proteoglycan aggregate isolated from Swarm rat chondrosarcoma. A central parameter that describes the kinetics of both types of water movement is the hydrodynamic frictional coefficient of water with proteoglycan. The frictional coefficient is markedly concentration dependent, increasing with increasing concentration, and highlights important structural features and types of organization of the proteoglycans in concentrated solutions. These include the requirements that proteoglycans in the extracellular matrix not to be immobilized but to have translational diffusive mobility and concentration gradients to be osmotically active, that chondroitin sulfate segmental mobility describing translational motion largely determines osmotic flow and hydraulic permeability of the proteoglycans, and that the proteoglycans exhibit an enhanced ability to resist flow as compared to other macromolecules. Additional dynamic studies suggest the formation of transient super-aggregate structures may occur at high concentrations which endows the proteoglycan subunit hydrodynamic properties similar to proteoglycan aggregate.     

Characterization of a proteinase inhibitor isolated from the fungal pathogen Coccidioides immitis.

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.     

Electrolyte distribution and active salt uptake in frog skin

1. The "chloride space" in frog skin was determined and found to be 69.7 per cent by weight of wet skin. The chloride space occupies about 94 per cent of the total water space of skin. From this and other information, it appears that the "non-chloride space" measures only a part of the space occupied by the structural elements of skin. This space is referred to here as the intracellular compartment and the remainder as the extracellular compartment of frog skin. On this basis, potassium and sodium in skin are distributed as follows: total sodium, 60 to 75 µeq./gm. of wet skin; all sodium is probably extracellular; total potassium, 39 to 49 µeq./gm.; intracellular potassium, 37 to 47 µeq./gm. 2. Skins were immersed in solutions differing from each other in their sodium and potassium concentrations. Three levels of NaCl were studied: 48, 119, and 169 µeq./ml. For each of these solutions (referred to below as diluted, physiological, and concentrated saline), the potassium levels were varied from 0.1 to 20 µeq./ml. For skins in solutions low in potassium and high in sodium, it was found that an exchange of intracellular potassium against extracellular sodium occurs. The ratio for the number of potassium ions lost/number of sodium ions gained was 4:1,4:6, and 4:8 for skin in K⁺-free diluted, physiological, and concentrated saline, respectively. 3. Uptake of NaCl by the epithelium of frog skin is dependent on the potassium concentration of the environment. For skins in physiological saline, net uptake of NaCl was optimal (0.90 µeq. x cm.^–2 x hr.^–1) at 1 to 5 µeq. K₊/ml. For skins in diluted and concentrated saline optimal NaCl uptake was seen at potassium concentrations of approximately 5 and 10 µeq. K₊/ml., respectively. Net uptake of NaCl by the skin is also discussed, with relation to the potassium balance of skin. 4. Skin potentials decreased with increasing extracellular potassium concentration when diluted saline solutions were used. The opposite of this was found for skins in concentrated saline. For skins in physiological saline, skin potentials rose sharply from rather low values, when placed in solutions very low in potassium, to relatively high values, when immersed in solutions containing 1 to 5 µeq. K⁺/ml. Further increase in potassium concentration of the bath led to slight reductions in skin potentials. The highest potentials observed were of the order of 40 mv. In all cases studied, the inside was positive with relation to the outside. 5. It can be shown that values for intracellular potassium concentration as a function of extracellular potassium concentration satisfy, at a first but good approximation, Freundlich's isotherm. A modification of Freundlich's isotherm, recently introduced by Sips, may also be used to correlate the experimental data quantitatively. Since the latter isotherm has a rational interpretation, it is suggested that this be used, rather than Freundlich's isotherm, to express quantitatively the dependence of intracellular on extracellular potassium in frog skin.     

高密度发酵和真空冷冻干燥工艺对乳酸菌抗冷冻性的影响

经真空冷冻干燥得到的乳酸菌发酵剂存活率和后期的低温贮藏稳定性与诸多因素相关。本文综述了制备乳酸菌发酵剂过程中高密度发酵和真空冷冻干燥工艺的不同对乳酸菌抗冷冻性的影响,其中高密度发酵过程中的培养基组分、培养温度、发酵恒定pH、中和剂的选择、菌体收获时期和发酵结束后处理以及真空冷冻干燥过程中保护剂的添加、预冷冻处理等是影响乳酸菌抗冷冻性的重要因素。通过对这些相关因素的综述分析,为提高乳酸菌发酵剂的冻干存活率和后期的低温贮藏稳定性提供新的思路,且应用抗冷冻性强、活力高的乳酸菌发酵剂对有效提高乳制品的质量和企业的经济效益意义重大。     

Cell water permeability and cryotolerance of Saccharomyces cerevisiae

Electron particle sizing (Coulter counter) was used to measure cell and protoplast volumes of Saccharomyces cerevisiae grown under different conditions designed to increase its cryotolerance. Membrane water permeabilities were estimated from those measurements. A relationship was obtained between the lower water permeability of yeast grown under microaerobic batch conditions and its weaker cryotolerance in water (cooling rate of 39·6°C/min), as compared to fed-batch cells. For the latter, cell water permeability was not related to the observed differences in survival for frozen-thawed cells grown under strong or partial (with temporary limitation of dissolved oxygen in growth media) aerobic conditions.     

Protein-protein association in polymer solutions: from dilute to semidilute to concentrated

In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor protein (BLIP) in the presence of crowding agents of varying molecular mass, from monomers (ethylene glycol, glycerol, or sucrose) to polymeric agents such as different polyethylene glycols (PEGs, 0.2-8 kDa) and Ficoll. An inverse linear relation was found between translational diffusion of the proteins and viscosity in all solutions tested, in accordance with the Stokes-Einstein (SE) relation. Conversely, no simple relation was found between either rotational diffusion rates or association rates (k(on)) and viscosity. To assess the translational diffusion-independent steps along the association pathway, we introduced a new factor, alpha, which corrects the relative change in k(on) by the relative change in solution viscosity, thus measuring the deviations of the association rates from SE behavior. We found that these deviations were related to the three regimes of polymer solutions: dilute, semidilute, and concentrated. In the dilute regime PEGs interfere with TEM-BLIP association by introducing a repulsive force due to solvophobic preferential hydration, which results in slower association than predicted by the SE relation. Crossing over from the dilute to the semidilute regime results in positive deviations from SE behavior, i.e., relatively faster association rates. These can be attributed to the depletion interaction, which results in an effective attraction between the two proteins, winning over the repulsive force. In the concentrated regime, PEGs again dramatically slow down the association between TEM and BLIP, an effect that does not depend on the physical dimensions of PEGs, but rather on their mass concentration. This is probably a manifestation of the monomer-like repulsive depletion effect known to occur in concentrated polymer solutions. As a transition from moderate to high crowding agent concentration can occur in the cellular milieu, this behavior may modulate protein association in vivo, thereby modulating biological function.     

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Long-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells.