The Exchange of sodium Ions in the Root of Zea mays

The apparent cation-exchange capacity of maize roots has beenestimated in 100 mM NaCL and I mM NaCl by observing the effluxkinetics of isotopically labelled sodium from this tissue. Thisapproach indicated that the apparent capacity was 70 m.equiv/kgof tissue (fresh weight) and 2 m.equiv/kg of tissue (fresh weight)in 100 mM and I mM NaCl respectively; the pH of the medium was5.8 in each case.Similar sodium efflux experiments were performedon isolated stelar tissue from these roots and such experimentsgave an apparent exchange capacity of 86 m.equiv/kg of tissue(fresh weight) and 6 m.equiv/kg of tissue (fresh weight) in100 mM and I mM NaCl respectively, both at pH 5.8.The dependenceof the magnitude of the apparent exchange capacity upon theelectrolyte concentration of the external medium is discussed.     

Effect of the removal of the COOH-terminal region of bacteriorhodopsin on its light-induced H+ changes.

Removal of the COOH-terminal region of bacteriorhodopsin by digestion with trypsin or papain reduces the yield of light-induced H+ release by 50-70%. The rate of H+ release is not affected significantly, but the half time of H+ uptake increases almost twofold. However, there is no effect on the photocycle of bacteriorhodopsin as judged by the yield and decay kinetics of the M412 photointermediate. The H+:M ratio in enzyme-digested membranes is approximately 0.4-0.8, whereas untreated membranes have a H+:M ratio of approximately 2. Purple membrane sheets stored in distilled water at 4 degrees C for prolonged periods also have a low H+:M ratio, probably due to protease activity associated with bacterial contamination. Electrophoresis on sodium dodecylsulfate-polyacrylamide gels showed that both the enzyme-treated and the stored purple membrane samples have a higher electrophoretic mobility compared to the fresh preparation. The reduction in molecular weight can be accounted for by the loss of several residues from the COOH-terminal portion of the bacteriorhodopsin. We propose that the COOH-terminal region is partially responsible for the high yield of H+ release by the purple membrane.     

The low affinity binding site for the cocaine analog, WIN 35,428 is an artifact of freezing caudate tissue.

Binding of the cocaine analog [3H] WIN 35,428 was investigated in rat and rabbit caudate. In membranes prepared from fresh tissue, [3H] WIN 35,428 binding was characterized by a single high affinity site with a Kd of 2.5 nM for the rabbit and 5.3 nM for the rat. In contrast, [3H] WIN 35,428 binding to membranes prepared from frozen tissue (stored at -70 degrees C) revealed two binding sites, a high affinity site similar to the one observed in membranes from fresh tissue and a low affinity site with a Kd of 39 nM for the rabbit and 65 nM for the rat. The low affinity WIN 35,428 binding site was observed only in membranes derived from frozen tissue, suggesting that it was an artifact produced by the freezing/thawing process.     

Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

Effects of storage on the distribution of high density lipoprotein subfractions in human sera

The current growing interest in the separation of the high density lipoprotein (HDL) subfractions suggested a comparative analysis of the HDL2 and HDL3 distribution in fresh and stored serum samples. Human sera were processed by rate zonal ultracentrifugation in a swinging bucket rotor, immediately after collection and after a 7-day storage at 4 degrees C, both in the presence and in the absence of 5.1 M sodium bromide. Samples stored in absence of salt show a marked decrease of the HDL3 mass, a reduction of its flotation rate, and significant changes in the lipid composition. The HDL2 concentration and composition are not altered by storage. The reported findings indicate that significant HDL3 modifications may occur in serum samples stored at 4 degrees C; these changes can be prevented by the addition of high concentrations of salt before storage.     

Changes in properties of the inner mitochondrial membrane during mitochondrial biogenesis in aging sweet potato tissue slices in relation to the development of cyanide-insensitive respiration.

Protein and phospholipid of the inner mitochondrial membrane in sweet potato root tissue increased after a lag phase during aging of the sliced tissue. The protein, but not the phospholipid, from aged slices was more insoluble in a solution containing sodium deoxycholate and sodium cholate than that from fresh tissue. There were differences in polypeptide composition between deoxycholate-cholate-soluble and -insoluble fractions as determined by polyacrylamide-gel electorophoresis of membrane fragments in the presence of sodium dodecyl sulfate. However, no difference was observed between mitochondrial membranes from fresh and aged slices. When disrupted mitochondrial membrane was subjected to equilibrium density centrifugation, two bands were obtained from aged slices but only one band from fresh tissue. The lighter band from aged slices was indentical to the single band from fresh tissue. The denser band was very poor in phospholipid, and the protein was very insoluble in deoxycholate-cholate solution. The denser membrane fragments possessed a cyanide-insensitive respiratory chain whereas the lighter did not. It is proposed that the development of the cyanide-insensitive respiration in aging slices is related to the formation of phospholipid-deficient mitochondrial membrane.     

Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach

In the bovine, as in many mammalian species, sperm are temporarily stored in the oviduct before fertilization by binding to the oviduct epithelial cell apical plasma membranes. As the oviduct is able to maintain motility and viability of sperm and modulate capacitation, we propose that proteins present on the apical plasma membrane of oviduct epithelial cells contribute to these effects. To verify this hypothesis, the motility of frozen-thawed sperm was determined after incubation for 6 h with purified apical plasma membranes from fresh or cultured oviduct epithelial cells or from bovine mammary gland cells as a control. Analysis of intracellular calcium levels was performed by flow cytometry on sperm incubated with fresh membranes using Indo-1 to assess the membrane effect on intracellular calcium concentration. The coculture of sperm with fresh and cultured apical membranes maintained initial motility for 6 h (65% and 84%, respectively). This effect was significantly different from control sperm incubated without oviduct epithelial cell apical membranes (23%), with mammary gland cell apical membranes (23%), or with boiled epithelial cell apical membranes (21%). Apical membranes from oviduct epithelial cells diminished the percentage of sperm that reached a lethal calcium concentration over a 4-h period (18.7%) compared with the control (53.8%) and maintained lower intracellular calcium levels in viable sperm. These results show that the apical plasma membrane of bovine oviduct epithelial cells contains anchored proteinic factors that contribute to maintaining motility and viability and possibly to modulating capacitation of bovine sperm.     

The ultrastructure of the uredospore of Puccinia recondita

Dry, fresh uredospores ofPuccinia recondita that have been killed by infra-red radiation showed no striking ultrastructural differences from dry, fresh, viable uredospores. Numerous spherical and elipsoidal mitochondria with distinct and deep cristae, ribosomes, endoplasmic reticulum and convoluted plasmalemma were well shown by both. When moistened and incubated, killed uredospores lost ultrastructural organization, whereas moistened, viable, fresh uredospores imbibed moisture, became more spherical and germination commenced. A more or less centrally located nucleus was seen with the double membrane invaginated at some points. The advance of the germ tube was initially by enzymic degradation and concluded by mechanical disruption of the degraded pore plug. The mitochondria and the endoplasmic reticulum increased in number and the stored lipid bodies were gradually depleted as germination progressed. Features known as the ‘foamy cytoplasm’ and ‘folded membranes’ were seen in the germinating uredospores only. It was suggested that the ‘foamy cytoplasm’ could be functionally similar to the glyoxisome because of the close association of the former to lipid bodies. The ‘folded membranes’ may be accumulated endoplasmic reticulum being transported to the site of wall formation.     

Reevaluation of the role of auxin binding site II

Binding of 1-naphthylacetic acid (1-NAA) was assayed in microsomal membranes from Zea mays coleoptiles and from hypocotyls of Cucurbita pepo. Auxin binding site II was differentiated from site I binding by using phenylacetic acid (PAA) to saturate site I binding capacity. The amount of type-II binding sites, per gram original fresh weight, was 34 pmol with Zea and 6.4 pmol with Cucurbita. When maize membranes were separated by dextran gradient centrifugation, auxin binding site II migrated coincident with tonoplast marker enzymes. The physiologically active auxin 4-chloroindoleacetic acid (4-Cl-IAA) competed very poorly with 1-NAA binding to both site I and site II. This result suggests that sites I and II are not involved in the regulation of growth. When comparing isolated outer epidermis with intact coleoptile of Zea, similar amounts and ratios of site I and site II binding activities were observed.     

Biofouling of reverse osmosis membranes used in river water purification for drinking purposes: analysis of microbial populations

Biofouling in water treatment processes represents one of the most frequent causes of plant performance decline. Investigation of clogged membranes (reverse osmosis membranes, microfiltration membranes and ultrafiltration membranes) is generally performed on fresh membranes. In the present study, a multidisciplinary autopsy of a reverse osmosis membrane (ROM) was conducted. The membrane, which was used in sulfate-rich river water purification for drinking purposes, had become inoperative after 6 months because of biofouling and was later stored for 18 months in dry conditions before analysis. SSU rRNA gene library construction, clone sequencing, T-RFLP, light microscope, and scanning electron microscope (SEM) observations were used to identify the microorganisms present on the membrane and possibly responsible for biofouling at the time of removal. The microorganisms were mainly represented by bacteria belonging to the phylum Actinobacteria and by a single protozoan species belonging to the Lobosea group. The microbiological analysis was interpreted in the context of the treatment plant operations to hypothesize as to the possible mechanisms used by microorganisms to enter the plant and colonize the ROM surface.     

Biofouling of reverse osmosis membranes used in river water purification for drinking purposes: analysis of microbial populations

Biofouling in water treatment processes represents one of the most frequent causes of plant performance decline. Investigation of clogged membranes (reverse osmosis membranes, microfiltration membranes and ultrafiltration membranes) is generally performed on fresh membranes. In the present study, a multidisciplinary autopsy of a reverse osmosis membrane (ROM) was conducted. The membrane, which was used in sulfate-rich river water purification for drinking purposes, had become inoperative after 6 months because of biofouling and was later stored for 18 months in dry conditions before analysis. SSU rRNA gene library construction, clone sequencing, T-RFLP, light microscope, and scanning electron microscope (SEM) observations were used to identify the microorganisms present on the membrane and possibly responsible for biofouling at the time of removal. The microorganisms were mainly represented by bacteria belonging to the phylum Actinobacteria and by a single protozoan species belonging to the Lobosea group. The microbiological analysis was interpreted in the context of the treatment plant operations to hypothesize as to the possible mechanisms used by microorganisms to enter the plant and colonize the ROM surface.     

Prolactin binding in the developing rat fetal liver

The binding of prolactin by fetal rat liver cell membrane fractions from 17 to 21 days gestation was studied. Particulate liver membranes were prepared in Dulbecco's Phosphate Buffered Saline (PBS) by ultracentrifugation and incubated at 22 degrees C for 16 hours with [125I] iodo-human growth hormone (hGH). Non-specific binding was assessed by parallel incubations in the presence of a 2000-fold excess ovine prolactin. Specific prolactin binding sites were detected only at 21 days gestation (2932 +/- 401 cpm/mg protein) in freshly prepared membranes. On freezing at -20 degrees C for 24 to 48 hours, the membranes of 20 days gestation animals were able to specifically bind prolactin (1295 +/- 239 cpm/mg protein). Freezing led to a 45 +/- 7% increase (4270 +/- 701 cpm/mg protein) in prolactin binding at 21 days gestation. No hormonal binding was detected from 17 through 19 days gestation in either fresh or freeze-thawed membranes. Scatchard analysis revealed a high affinity binding site with a Ka of approximately 1.4 X 10(8)M-1 in both fresh and freeze-thawed membrane preparations. The data show that 1) prolactin receptors appear in liver only during late fetal life and that 2) freezing of membranes may unmask binding sites that are initially unavailable to specifically bind prolactin.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Comparative studies on the polypeptide composition of chloroplast lamellae and lamellar fractions

The polypeptide composition of spinach chloroplast membranes and membrane fractions has been examined by the technique of sodium dodecylsulfate-polyacrylamide gel electrophoresis. Chloroplasts were fragmented into grana (Photosystem II enriched) and stroma lamellae (Photosystem I in character) by the French press technique. The grana lamellae were futher fractionated by the use of digitonin into two fractions, one enriched in Photosystem II and the other enriched in Photosystem I. These membranes are composed of at least 15 polypeptides two of which, with approximate weights of 39 and 50 kdaltons, are observed only in granal fractions. Quantitatively the primarily Photosystem II fractions are enriched in polypeptides in the 30-23 kdalton range whereas the Photosystem I (or Photosystem I-enriched) fractions are enriched in polypeptides in the 60-54 kdalton region. The experiments reported show that contamination by soluble proteins or other membranes is negligible. The results indicate that subtle differences in composition account for the large differences in structure and function within the chloroplast membrane system.     

Radiation Processed Amniotic Membranes in the Treatment of Non-Healing Ulcers of Different Etiologies

The amniotic membranes were collected from the placentae of selected and screened donors. Processing was done by washing the fresh amniotic membrane successively in sterile saline, 0.05% sodium hypochlorite solution and sterile distilled water until it was completely cleared of blood particles. The membranes were sterilized by gamma irradiation at 25 kGy. The processed amniotic membranes were applied to 50 open wounds comprising of 42 full thickness defects and eight partial thickness defects. These included leprotic, diabetic, traumatic, gravitational ulcers and superficial burn in the form of scald and corrosive burn. The radiation processed amniotic membranes favoured healing of unresponsive and non-healing ulcers of different etiologies. Ulcers with duration of minimum 3 weeks to maximum 12 months were found to heal in 2-6 weeks by the application of amniotic membranes.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Characterization of the electrogenic sodium channel from rat brain membranes using neurotoxin-dependent 22Na uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to 22Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED50 = 0.2 micrometer), veratridine (ED50 = 1 micrometer), or grayanotoxin I (ED50 = 30 micrometers) stimulated 22Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca2+ and in a partial or allosteric competitive manner by lidocaine and procaine. This 22Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.     

Effect of freezing on the coupling of VIP receptors to adenylate cyclase in rat liver membranes

In fresh rat liver plasma membranes, high affinity VIP receptors were specifically labelled with [125I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp (NH)p, NaF and glucagon stimulations.     

Microbial dynamics of commercial makgeolli depending on the storage temperature

Market fresh makgeolli was stored at different temperatures of 4°C and 25°C to assess the change of the microbial diversity according to the storage temperature and period. Yeast counts increased until day 3 of storage and decreased thereafter. General and lactic acid bacterial counts continuously increased during storage. The data indicated that the control of growth of microorganisms, particularly general bacteria and lactic acid bacteria (LAB), is essential. Total acid levels started to decrease in the makgeolli stored at 4°C, and increased from day 6 of storage in the makgeolli stored at 25°C. The increase of total acid in the non-refrigerated condition greatly affected the quality of makgeolli. In both the fresh makgeolli samples stored at 4°C and 25°C, yeast (Saccharomyces cerevisiae) and molds (Aspergillus tubingensis, Candida glaebosa, and Aspergillus niger) were noted. Denaturing gradient gel electrophoresis (DGGE) band patterns were almost constant regardless of the storage period. As for bacteria, Lactobacillus crustorum, L. brevis, and Microlaena stipoides were found in the makgeolli stored at 4°C, and L. crustorum, Lactobacillus sp., L. plantarum, L. brevis, L. rhamnosus, and L. similis were found in the makgeolli stored at 25°C. In particular, in the makgeolli stored at 25°C, L. crustorum and L. plantarum presented dark bands and were identified as the primary microorganisms that affected spoilage of fresh makgeolli.