Membrane alterations associated with “transformation” by BUdR in BUdR-dependent cells : Fluorescence polarization studies with a lipid probe

Steady state and nanosecond fluorescence polarization studies were carried out on membranes of a “bromodeoxyuridine (BUdR) dependent” cell line (B4) derived from a malignant Syrian hamster melanoma line. When grown in the presence of BUdR B4 cells resemble transformed cells (in terms of several biological characteristics), while B4 cells grown in the absence of BUdR resemble untransformed cells. B4 cells were labelled with the lipid probe 1,6-diphenyl-1,3,5-hexatriene, which had been used previously to show that fluorescence polarization values of membrane lipids of virally transformed cells are higher than fluorescence polarization values of membrane lipids of untransformed cells. The steady state fluorescence polarization values of membrane lipids of B4 cells in BUdR were found to be larger than those of cells in the absence of BUdR, and the change in fluorescence polarization values was found to be fully reversible. Nsec rotational correlation time experiments confirmed and extended the steady state results. The results of the fluorescence polarization studies suggest that the membranes of B4 cells grown in the presence of BUdR resemble those of virally transformed cells while membranes of B4 cells grown in the absence of BUdR resemble those of untransformed cells.     

Fusion with enucleated fibroblasts corrects “I-cell” defect

Fusions have been carried out between fibroblasts from patients with “I-cell” disease and enucleated human fibroblasts with a single lysosomal enzyme deficiency derived from patients with G_M1-gangliosidosis, Sandhoff disease and mannosidosis. Pure cytoplasts were obtained using cytochalasin B treatment followed by fluorescence activated cell sorting. After fusion with whole “I-cells”, the cybrid populations showed a restoration of deficient lysosomal enzyme activity and also the abnormal electrophoretic pattern characteristic for the residual hexosaminidase activity in “I-cells” was found to be corrected. The results described in this paper indicate that the defective post-translational modification, which is responsible for the multiple lysosomal enzyme deficiency, can be corrected by a factor that is stable for at least three days in enucleated cells. During this period the cytoplasmic factor can act without the need of de novo synthesis but the absence of correction in in vitro experiments shows that cellular integrity is required.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

“Harpoon” Model for Cell–Cell Adhesion and Recognition of Target Cells by the Natural Killer Cells

The mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition or NK-sensitive target cells (TCs) by NK cells of NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of “non-NK receptors” and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.); (2) the presence of multiple disorders in the organization of “extracellular matrix-surface membrane-submembrane cytoskeleton” assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can “harpoon” the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this “harpoon” model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.     

Protein synthesis during fetal development of mouse epidermis : I. The appearance of “histidine-rich protein”

The synthesis of specific proteins at different stages in the development of mouse epidermis was investigated. The epidermis from mice of an age range from 16 days of fetal life to 7 weeks postpartum was prepared by maceration in acetic acid, and the total proteins were separated by SDS-polyacrylamide gel electrophoresis. The appearance of particular proteins at different developmental stages was correlated with the morphology of the epidermis at the same stage. Several qualitative and quantitative changes in the protein patterns were observed during development, the significance of which is discussed. A protein of molecular weight approximately 27,000 was the major component of a group of closely related proteins which appeared simultaneously with the production of mature keratohyalin granula in 18-day fetal mouse epidermis. Isolation and characterization of this protein showed it to be a “histidine-rich protein.” Its concentration in the epidermis decreased as the number of keratohyalin granula diminished with increasing age. The protein could be localized exclusively in the keratohyalin granula by a fluorescence method involving staining with dansyl chloride. This method was also used to detect “histidine-rich protein” on polyacrylamide gels.     

Réoxydation du quencher de fluorescence “Q” en présence de 3-(3,4-dichlorophényl)-1,1-diméthylurée

Reoxidation of the fluorescence quencher “Q” in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Reoxidation of the fluorescence quencher Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea shows the following properties:

It is sensitive to very low concentrations of hydroxylamine (a few μM).

It corresponds to a back reaction between Q⁻ and the primary oxidant Z⁺ formed in the light. A part of this back reaction gives rise to luminescence emission.

Within the range we studied the kinetic of reoxidation is second order with regards to Q⁻.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

Hydrology of winter–spring “red tides” in Bahía de Mazatlán, Sinaloa, México

“Red tide” events are frequent and periodical in Bahía de Mazatlán, Sinaloa, México. Yet, the ones observed from 4 February to 4 June 2000, showed some distinctive features: First, the dinoflagellates Prorocentrum balticum (85%), P. mexicanum (5%), and Ceratium furca (5%), dominated the composition of the blooms; Second, the average cell abundance by date was 1.3×10⁶ cells l⁻¹, with a range of 3.5×10³ to 24,500 × 10³ cells l⁻¹, well above previous records; Third, the temperature registered at 10–20 m deep was unusually cold (19 °C), below the normal average of 21.5 °C observed over the last 10 years. Salinity was high (35.9 psu) and showed very little influence on the water density gradient. A mean thermal stratification index (TSI), of 3.4, with a maximum of 7 °C, was observed throughout the period, in spite of a weak upwelling activity and intermittent strong NW winds which were unable to break up water column stratification. Temperature fluctuations at the surface and at the bottom layers showed a 30-day periodicity, suggesting some association with the lunar cycle. To explain the characteristics of the “red tides” registered in Bahía de Mazatlán during the winter–spring period of year 2000, it is proposed that the temperature and density stratification, stabilized further by internal waves that compensated for the weak upwelling activity and provided the necessary nutrients to the surface layer, favored the persistence and intensity of the harmful algal bloom events then observed.     

Clonal selection by fluorescence redistribution after photobleaching (FRAP)—A “fast” lateral mobility fibroblast mutant (E7G1)

Fluorescence redistribution after photobleaching (FRAP) was utilized to select a “fast” lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 ± 1.6) × 10⁻⁹ cm²/s and (2.7 ± 2.3) × 10⁻⁹ cm²/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 ± 1.9 × 10⁻⁹ cm²/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 ± 0.19) × 10⁻¹⁰ cm²/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.     

A long-wave absorbing form of chlorophyll a responsible for the “red drop” in fluorescence at 298 °K and the F723 band at 77 °K

When Chlorella cells are ruptured at pH 4.6 by sonication in air, its absorption spectrum can be best explained if one assumes that a long-wave chlorophyll a form (Chl a 693) is preferentially destroyed. Using these preparations, and comparing them with the algal suspension and the sonicates prepared at pH 7.8 under argon, we make the following conclusions: (a) The red drop beginning at about 675–680 nm in the action spectrum^* of fluorescence at 298 °K must be due to the presence of a non-(or weakly) fluorescent form of chlorophyll a. We suggest that this form is Chl a 693. The red drop is absent in the aerobic sonicates. (b) The red drop in fluorescence in whole algal cells is not due to any errors in absorption measurements; this drop is clearly present in the anaerobic sonicates. (c) The emission band at 723 nm, discovered by in whole Chlorella cells at 77 °K, may be due to increased fluorescence efficiency of Chl a 693 at low temperature; the F723 band is absent in aerobic sonicates.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

The “erythromycin-resistance” methylated sequence of Staphylococcus aureus ribosomal RNA

We have determined the sequence of an oligonucleotide from the large ribosomal subunit RNA of Staphylococcus aureus whose methylation renders the organism resistant to erythromycin and other antibiotics (the “MLS” phenotype). Analysis of RNase A digests of [³H]methyl-, ³²P-labeled RNA yielded the sequence GG · m₂⁶A · AAGACp, where m₂⁶A is an N⁶-dimethylated adenosine residue that in sensitive cells is unmethylated. Comparison with homologous sequences recently reported for Saccharomyces cerevesiae mitochondria indicates that an A to G mutation in this latter system mimics dimethylation in St. aureus with regard to functional consequences.     

Development of the transgenic cyan fluorescent protein (CFP)‐expressing nude mouse for “Technicolor” cancer imaging

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non‐transgenic nude mice with the transgenic CK/ECFP mouse in which the β‐actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA‐1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color‐coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color‐coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. J. Cell. Biochem. 107: 328–334, 2009. © 2009 Wiley‐Liss, Inc.     

Molybdenum EXAFS of the desulfovibrio gigas MO(2Fe-2S) protein—structural similarity to “desulfo” xanthine dehydrogenase

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 Å) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand a 1.90 Å. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 Å contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with “desulfo” xanthine oxidase.     

“Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan under acidic conditions: A rheological and calorimetric study of the mechanism of synergistic gelation

The effect of acidification on a typical commercial xanthan and on pyruvate-free xanthan (PFX), alone and in gelling mixtures with konjac glucomannan (KGM), has been studied by differential scanning calorimetry (DSC) and small-deformation oscillatory measurements of storage modulus (G′) and loss modulus (G″). For both xanthan samples, progressive reduction in pH caused a progressive increase in temperature of the disorder–order transition in DSC, and a progressive reduction in gelation temperature with KGM. This inverse correlation is interpreted as showing that synergistic gelation involves disruption of the xanthan 5-fold helix, probably by attachment of KGM to the cellulosic backbone of the xanthan molecule (as proposed previously by a research group in the Institute of Food Research, Norwich, UK). Higher transition temperature accompanied by lower gelation temperature for PFX in comparison with commercial xanthan at neutral pH is explained in the same way. However, an additional postulate from the Norwich group, that attachment of KGM (or galactomannans) can occur only when the xanthan molecule is disordered, is inconsistent with the observation that gelation of acidified mixtures of KGM with PFX can occur at temperatures more than 60 °C below completion of conformational ordering of the PFX component (as characterised by DSC). Increase in G′ on cooling for mixtures of commercial xanthan with KGM at pH values of 4.5 and 4.25 occurred in two discrete steps, the first following the temperature-course observed for the same mixtures at neutral pH and the second occurring over the lower temperatures observed for mixtures of KGM with PFX at the same values of pH. These two “waves” of gel formation are attributed to interaction of KGM with, respectively, xanthan sequences that had retained a high content of pyruvate substituents, and sequences depleted in pyruvate by acid hydrolysis. At pH values of 4.0 and lower, gelation of mixtures of KGM with commercial xanthan followed essentially the same temperature-course as for mixtures with PFX, indicating extensive loss of pyruvate under these more strongly acidic conditions. Mixtures prepared at pH values in the range 4.0–3.5 gave comparable moduli at room temperature (20 °C) to those obtained at neutral pH, but showed substantial softening on heating to body temperature, suggesting possible applications in replacement of gelatin in products where “melt-in-the-mouth” characteristics are important for acceptability to the consumer.     

“Menstrual induction” with sulproston

The PGE₂-analogue Sulproton (16-phenoxy·ω-17,18,19,20-tetranor-PGE₂-mythylsulfonylamide) was administered to 200 medically and gynecologically normal women who were 17±0.4 days beyond their expected menstrual period and who had a positive pregnancy test. The intramuscular impact dose (500 μg repeated after 4 hours) caused an immediate tonic uterine contraction which compromised the estradiol 17β, progesterone and chorionic gonadotropin production within the fetoplacental unit, and thereby allowed the evolution of cyclic uterine activity, cervical dilatation and tissue expulsion.Pregnancy termination was complete in 92% of women, 5.5% required surgical curettage and 2.5% were given a second Sulproston treatment 2–3 weeks after the first to remove retained tissue from the uterus. The medical induction of menstruation was preferred by 83% of the women who had previously experienced surgical termination of pregnancy. Normal menstruation resumed in all women after 36±0.9 days. The majority of 42 women questioned found Sulproston a satisfactory, safe, simple and effective drug regimen for “menstrual induction”.     

The Stability Of So-Called Axonal Acid Phosphatase As Determined By Experiments In Its “Stainability”

Since the histochemical method for exhibiting acid phosphatase in bodily tissues is said to depend upon the enzyme acting on suitable substrates, it is possible to test its stability by various tests. It has been found that the background element or elements, whatever they may b e, concerned with the “staining” properties of the reaction are very stable and somewhat resistant to destruction. So-called acid phosphatase in spinal cord axons has not been inactivated by subjecting it to various fixing solutions, changes in temperature and pH, relatively prolonged post-mortem autolysis nor by well known enzyme inactivators. It is believed that the “staining” reaction may be dependent fundamentally on other factors than enzymatic activity.