Structural reorganizations of the lipids and proteins of erythrocyte membranes under the action of low temperatures

The reversible structural rearrangement of lipids and protein oligomerization has been shown to occur during cooling in membranes of model systems (liposome, erythrocyte shadows) and native erythrocytes. Analysing the dependence of Azz in membrane probes (5- or 15-doxylstearic acids) in the Arrhenius plots a conclusion on the structural changes at 13-19 degrees C and within the range of interior water freezing from -17 up to -19 degrees C has been drawn, the last transition is smoothed out in the presence of glycerin. Using diamide joining spectrin and electrophoresis in polyacrylamide gel it has been determined that the low temperatures cause the spatial approach of proteins of spectrin-actinic complex and formation connections between the erythrocyte membrane proteins which aren't destroyed by dodecylsulfate.     

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.     

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

Isolation and characterization of the K6 type yeast killer protein

The optimum production of K6 type yeast killer protein by Kluyveromyces fragilis NCYC 587 occurred at pH 4.0-4.4 and at 22-24 degrees C in a killer-zone assay test. The K6 killer protein was concentrated by acetone precipitation of the culture supernatant and purified by native polyacrylamide rod gel electrophoresis. The protein migrated as a single band on discontinuous gradient SDS polyacrylamide gel electrophoresis and had a molecular weight of 42,313. The isoelectric point of the K6 type protein was determined at pH 5.97 by high voltage vertical polyacrylamide gel electrofocusing. Western blot analysis revealed that the K6 killer toxin was a nonglycosylated protein.     

Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures

The heat stress response was studied in Lactobacillus helveticus PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20 degrees C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42 degrees C. Proteinase, peptidase, and acidification activities of L. helveticus PR4 were found to be higher in cells harvested when 40 degrees C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42 degrees C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55 degrees C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20 degrees C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42 degrees C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40 degrees C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.     

Preparation and purification of polymerized actin from sea urchin egg extracts

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F- actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.     

Binding of lithium dodecyl sulfate to polyacrylamide gel at 4 degrees C perturbs electrophoresis of proteins

Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25 degrees C, the gel maximally binds 17 mg of LDS per gram dry weight at 4 degrees C. When polyacrylamide gel electrophoresis is carried out at 4 degrees C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4 degrees C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25 degrees C.     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.     

Varying patterns of protein synthesis in bread wheat during heat shock

High temperatures during seedling growth are considered as one of the factors that can modify surviving properties in wheat (Triticum aestivum L.) plant. This work attempts to evaluate the heat shock responses of seedling of winter wheat (Bezostaya-1) using growth parameters (seedling length, embryonal root length and embryonal root number), membrane stability index (MSI) and two dimensional (2D) gel electrophoresis analysis of heat shock proteins (HSPs) during heat shock. Seedlings grown until first leaf opening at controlled conditions (23 degrees C, 200 micromol m(-2) s(-1), 16h day/8h night, 50-60% humidity) were exposed to 37 degrees C or 45 degrees C high temperatures for 2, 4 and 8 hours. While 37 degrees C did not cause any significant change, 45 degrees C heat treatments caused significant decrease in terms of seedling and root length, and leaf MSI for all exposure times. However, all the plants from 45 degrees C heat treatments continued to grow during recovery period. 2D protein analysis indicated that 37 degrees C, 8 hours exposure caused stronger and more diverse heat shock response than the other treatments, followed by 37 degrees C, 4 hours, 45 degrees C, 8 hours, 45 degrees C, 4 hours, 45 degrees C, 2 hours treatments. 5 protein spots, ranging from 6-7.8 pl (isoelectric point) and 27-31.7 kDA molecular weight, were expressed at 37 degrees C, 2 hours and continued at 37 and 45 degrees C for all exposure times. This suggests that these early proteins and other newly synthesized proteins may have protective effects at 37 and 45 degrees C and provide plants for healthy growth during the recovery period.     

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

Lung surfactant (LS) is an extra-cellular lipid-protein system responsible for maintaining low surface tension in the lung and alveolar stability. Serum proteins cause dysfunction of this material, e.g. in adult respiratory distress syndrome (ARDS). BLES is a clinically used LS consisting of most of the lipids and associated proteins from bovine lung lavage. Aqueous phases of BLES at 30% and 70% hydration, with and without 5% by weight of bovine serum albumin (BSA), calculated on the amount of lipids, were studied using X-ray diffraction during cooling from 42 to 5 degrees C. The diffraction curves are consistent with a transition from a lamellar liquid crystalline phase to a gel phase transition at cooling in the interval 30-20 degrees C. The long-spacings correspond to a reduction of the bilayer thickness during this transition. The wide-angle region shows a peak at 4.1 A below 25 degrees C, which is characteristic of the hexagonal chain packing of the gel phase. The perturbation of the bilayers by the presence of BSA seems to induce a significant decrease of the bilayer thickness. Calculations on the observed limits of swelling (taking place in the range 50-60%) indicate that BSA is closely associated with the BLES bilayers, probably due to electrostatic interaction with the cationic surfactant proteins SP-B and SP-C. This study show that the LS lipid structural organizations are extremely susceptible to small amounts of serum albumin, which may have implications in surfactant related lung disease and clinical applications of surfactant therapy.     

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Morphogenesis of Candida albicans and cytoplasmic proteins associated with differences in morphology, strain, or temperature.

The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, which produces yeast cells exclusively at both 24 and 37 degrees C in low-sulfate medium. The proteins of both strains were labeled at both temperatures with [35S]sulfate, cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation, and the labeled proteins were analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. Ten protein spots were identified on the two-dimensional gels of the 4918 mycelial-phase extract which were not present in the 4918 yeast-phase extract. These proteins appeared to be modifications of preexisting yeast-phase proteins rather than proteins synthesized de novo in the mycelial cells because 5 were absorbed by rabbit anti-yeast-phase immunoglobulin and each of the 10 was also present in extracts of strain 2252 grown at 24 and 37 degrees C, indicating that they were neither unique to filamentous cells nor sufficient for induction or maintenance of the mycelial morphology. Thirty-three proteins were identified in the 4918 yeast-phase extract which were not present in the 4918 mycelial-phase extract. Pulse-chase experiments revealed the synthesis of new proteins during yeast-to-mycelial conversion, but none of these was unique to mycelial cells. No differences in the major cytoplasmic proteins of any of the yeast- or mycelial-phase extracts were identified. This finding suggests that the major structural proteins of the cytoplasm are not extensively modified and argues instead that proteins unique to either phase may serve a regulatory function.     

Phosphorylation in coronary artery cold-induced contraction in the newborn lamb

Myocardial dysfunction after hypothermic protection has been linked to various mechanisms. Coronary vasospasm in particular may be responsible for ischemic injury during reperfusion. Herein we hypothesized that coronary arteries (CA) sustain a cold-induced contraction during hypothermia mediated by a protein tyrosine kinase (PTK)-/protein tyrosine phosphatase (PTP)-dependent pathway. Isolated newborn lamb CA rings were studied in a tissue bath for isometric contraction during 2-h profound (17 degrees C) or ultra-profound (7 degrees C) hypothermia. In parallel, protein tyrosine phosphorylation was evaluated by use of the Western blot technique. Na-orthovanadate (SOV) and genestein (GEN) were used separately and in combination to evaluate the effect of PTK/PTP activation on CA contraction and tyrosine phosphorylation during cooling (17 or 7 degrees C) vs 37 degrees C. Cooling from 37 to 7 degrees C induced transient contraction at approximately 17 degrees C (29% KCl response), which was more prominent during rewarming to 37 degrees C (36% KCl). Cooling to 17 degrees C resulted in sustained contraction (7-10% KCl), which was reversible upon rewarming. Cold-induced contraction was significantly enhanced by SOV (7- to 10-fold at 17 degrees C; 2-fold at 7 degrees C) and abolished by GEN. Concurrently, tyrosine phosphorylation of 33-, 45-, and 104-kDa proteins increased during cooling (35-100% at 17 degrees C; 46-66% at 7 degrees C). Tyrosine phosphorylation was similarly enhanced by SOV (1.7- to 2.3-fold at 17 degrees C; 2.9- to 3.9-fold at 7 degrees C) and abolished by GEN in the presence or absence of SOV. These results support a prominent role for the PTK/PTP signal transduction pathway in the coronary artery cold-induced contraction. This information provides one possible biomolecular mechanism linked to ischemia/reperfusion pathophysiology of CA in neonatal hearts exposed to hypothermic myocardial protection.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide)

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.     

Heat Stress Induces Changes in Protein Synthesis and Fast Axonal Transport in Bullfrog Sensory Neurons

The effects of heat stress on protein synthesis and fast axonal transport were examined in an in vitro bullfrog primary afferent neuron preparation. The magnitude of effect was determined for individual [35S]methionine-labelled protein species separated via two-dimensional gel electrophoresis. Elevation of temperature of the preparation from 18 degrees C to 33 degrees C caused a transient inhibition of synthesis of non-heat-shock proteins, whereas the synthesis of a 74,000-dalton protein increased to 927% of controls after 4 h. Similar prolonged stress conditions had no effect on the relative abundance of 36 individual, newly synthesized proteins undergoing fast axonal transport. A dramatic exception was represented by a 55,000-dalton glycoprotein whose fast transport was increased to 291% of control. The increase in transport of this protein during a time when synthesis and transport of other non-heat-shock proteins were not enhanced suggests that it may play a unique role in the early cellular events that mediate survival or thermotolerance in the neuron.