Rat erythrocyte insulin receptors: radioreceptor assay and characterization

Highly specific insulin receptors have been identified on the rat erythrocyte. A radioreceptor assay for the evaluation of these receptors has been developed, and the characteristics of these receptors have been investigated. Insulin receptor binding on the rat erythrocytes was found to be dependent on pH, temperature, time, and ionic strength. When incubated for 3½ hours at 15° C, 5.0 × 10⁹ erythrocytes/mL from each of 10 rats were found to bind specifically 7.54 percent (±0.15 SEM) of 40 pg of ¹²⁵I-insulin. Specific binding was found to be a function of cell concentration. The pH optima for insulin binding were found to be 7.4 and 7.0 in the absence of cations. The presence of cations not only shifted pH optimum to 7.4 from 7.0, but also increased specific insulin binding.     

Quartz-crystal microbalance-dissipation technique for the study of initial adsorption of fibronectin onto tresyl chloride-activated titanium

The immobilization of cell-adhesive proteins onto titanium implants improves biological response at the implant-tissue interface. Previous studies demonstrated the easy and direct attachment of fibronectin onto titanium with the use of a 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) activation technique. The present study investigated the initial adsorption behavior of fibronectin on tresyl chloride-activated titanium by the quartz-crystal microbalance-dissipation (QCM-D) technique. The crystal resonant frequency and the dissipation shift of the oscillator were simultaneously measured by the injection of fibronectin/phosphate-buffered saline solution (pH = 7.4). The tresyl chloride-activated titanium surface showed a faster and greater decrease in frequency than that of untreated titanium, indicating that a greater amount of fibronectin was adsorbed in the former case during a 120-min adsorption. The dissipation-frequency plots revealed that, during the initial stage of adsorption, the bond between fibronectin and tresyl chloride-activated titanium is stronger than that between fibronectin and untreated titanium. The QCM-D technique can provide new insights into the adsorption mechanism of fibronectin.     

Migration Inhibition Produced by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride

Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intraperitoneal injection of Marcol oil. The washed cells were exposed to various concentrations of sodium periodate in phosphate-buffered saline (PBS) at pH 7.4 for 10 min at +4 degrees C. The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10(-3) to 10(-5) M. The viability of the macrophages was not affected by this treatment. Sodium borohydride (10(-3) to 10(-5) M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate for 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition.     

Adorption of human fibrinogen and human serum albumin onto polyethylene

The adsorption of human fibrinogen (HFb) onto polyethylene at pH 7.4 and 37°C is irreversible with the saturated value 0.51 μg/cm². The established dependence of the concentration of irreversibly adsorbed HFb on the concentration of HFb in solution does not reflect an equilibrium between adsorption and desorption and cannot be described by an adsorption isotherm. No reversible adsorption of HFb and HSA at 37°C has been observed. At lower temperatures, both reversibly and irreversibly adsorbed HFb have been detected.     

Influence of volume dilution, lactate, phosphate, and calcium on mitochondrial functions.

Oxidative phosphorylation of isolated canine myocardial mitochondria has been evaluated after exposure to different concentrations of phosphate (5--50 mM), lactate ion in excess (5--40 mM, pH 7.4), calcium (50--270 nmol/mg protein), to lactic acidosis (pH 6.3), and to mitochondrial protein dilution (in vitro volume expansion) for 10 min to 8 h. The influence of phosphate and lactate ion addition, lactic acidosis, and in vitro volume expansion on mitochondrial function were studied in the isolation medium (0.18 M KCl, 0.5% BSA (bovine serum albumin), with or without Tris-EDTA, pH 7.4) prior to evaluation of mitochondrial function in the assay medium (0.25 M sucrose, 10 mM Tris-HCl, and 10 mM inorganic phosphate, pH 7.4). The effect of calcium addition was assessed in the assay medium. The results of these studies demonstrate that each of these interventions detrimentally alters mitochondrial oxidative phosphorylative ability. The most severe mitochondrial functional impairment resulted from phosphate or calcium addition. The detrimental effect of phosphate and in vitro volume expansion was partially corrected by the addition of cytochrome c.     

Evaluation of calcium titanate as apatite growth promoter

Calcium titanate (CaTiO(3), perovskite) has been used to determine its apatite nucleation ability and propose a possible nucleation initial step. Measurements of calcium leaching from the calcium titanate surface and phosphate adsorption experiments were carried out separately by using commercial calcium titanate suspensions at room temperature. Adsorption behaviour determined by zeta potential measurements shows that phosphate is strongly adsorbed on the calcium titanate surface. It was found that the higher the pH, the higher the Ca present on the calcium titanate surface, but phosphate adsorption followed this trend only up to pH 7.4. Results suggest that phosphate ions are not adsorbed only on Ca sites but also on TiO(2) groups sites of the surface, formed after calcium leaching from the surface. When both ions are simultaneously added in a modified simulated body fluid containing calcium titanate, at 37 degrees C, apatite growth occurs on its surface after 1 week of immersion.     

pH对体外培养成年大鼠嗅鞘细胞存活的影响

为了研究不同pH培养环境对体外培养大鼠嗅鞘细胞(OECs)存活的影响,本研究采用成年大鼠嗅球培养OECs,培养第8d用S-100免疫组化鉴定纯度后,改用不同pH值(6.8,7.0,7.4,7.8)的DF12培养液继续,在相差显微镜下观察OECs的形态学变化。在不同pH条件下继续培养第3d时采用MTT方法检测不同pH环境中的细胞活力,并用碘化丙啶(PI)和Ho-echst33342荧光双重染色观察细胞死亡率。结果显示:大鼠OECs纯度为(70±4)%;pH7.4组OECs生长旺盛,pH6.8组及pH7.0组OECs生长增殖受到不同程度的抑制,出现死亡细胞;pH7.8组OECs广泛死亡;MTT试验表明,pH7.4组的OD值显著高于其他各pH组。PI/Hoechst33342双重荧光染色显示,在pH6.8、pH7.0和pH7.8组死亡OECs的百分比分别为(27.6±7.8)%、(31.7±7.2)%和(62.3±5.6)%。在pH7.4组未见到死亡的OECs。研究结果表明OECs对酸、碱性环境改变均较敏感,尤其是碱性环境。     

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.     

The serology of the bromoviruses. I. Serological interrelationships of the bromoviruses

The serological relationships between brome mosaic (BMV), cowpea chlorotic mottle (CCMV), and broad bean mottle (BBMV) viruses, and their coat proteins, were studied by gel precipitin, "rocket immunoelectrophoresis" (RIE), and enzyme-linked immunosorbent assay (ELISA) techniques. Gel precipitin and RIE tests indicated that capsid swelling altered the antigenicity of both BMV and CCMV, as although the coat proteins were serologically related at both pH 6.0 and pH 7.0, the viruses appeared to be related only when swollen at pH 7.0. Fixation of the viruses with 2% formaldehyde at pH 6.0 appeared to remove the relationship. BBMV was not related by precipitin tests to either BMV or CCMV but was related to both by direct and indirect ELISA at both pH 6.0 and pH 7.4, though less closely than BMV and CCMV were related to each other. Indirect ELISA showed the three coat proteins to be more closely related than the parent viruses. Formalinized BMV and CCMV appeared less related than the native viruses at pH 6.0. Sandwich ELISA proved too strain specific to show any group relationships. The implications of these results and a serological map proposed for the bromoviruses are discussed.     

Reversible biotinylation of C1q with a cleavable biotinyl derivative Application in C1q receptor (C1qR) purification

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (M_r 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 × 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) ¹²⁵I-surface labeled membrane solution that had been first centrifuged (1 h, 45 000 × g, 4°C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4°C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major M_r 70 000 molecule which upon reduction electrophoresed with an apparent M_r of 85 000–90 000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30–40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.     

In vitro studies of the interaction of poly(NIPAm/MAA) nanoparticles with proteins and cells

The pH- and temperature-responsive poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAm/MAA) nanoparticles are of potential application in targeted drug delivery. Their responsive properties in the presence of human serum albumin were investigated using dynamic light scattering (DLS), protein assay, and electron spin resonance (ESR) spectroscopy. Their interaction with human monocytes and polymorphonuclear leukocytes (PMNLs) was studied using scanning electron microscopy (SEM) and oxygen consumption method. The nanoparticles exhibited a volume phase transition at 35-40 degrees C in Hanks balanced salt solution (HBSS) and in phosphate buffer solution (PBS) of pH 7.4. The diameter of the nanoparticles decreased slightly in the presence of HSA at 25 degrees C at neutral pH, whereas an increase in the diameter in pH 6 PBS at 40 degrees C was revealed. The amount of albumin adsorbed onto the nanoparticles decreased with increasing temperature. The ESR spectra of spin labeled HSA indicated a more restricted environment in the nanoparticles at elevated temperatures. The stimulation of PMNL oxygen consumption by PNIPAm based nanoparticles, an indication of phagocytosis of the particles, was not observed regardless whether the nanoparticles were incubated in plasma or serum. In contrast, the more hydrophobic polystyrene (PSt) particles induced a significant increase in the rate of oxygen consumption after the incubation. PNIPAm/MAA-grafted-PSt particles behaved similarly to the PNIPAm/MAA nanoparticles, suggesting that surface properties dictate the recognition of colloids by PMNLs.     

A one-step isolation procedure for phospholipase A2-free cobra venom factor by fast protein liquid chromatography

A very rapid and efficient procedure for isolation of cobra venom factor (CoF) from Naja naja venom is presented. The method is based on Mono Q anion exchange chromatography on a system for fast protein liquid chromatography (FPLC). CoF was eluted by a buffer of pH 7.4 at 280 mM salt. A purification of 33.7 X was reached with a yield of at least 27%. Contamination with phospholipase was under the detection limit of a sensitive radiometric assay (less than 25 ppm), while the starting material contained 5%. The preparation displays high C-depleting activity in vivo.     

In vitro studies of the interaction of poly(NIPAm/MAA) nanoparticles with proteins and cells

The pH- and temperature-responsive poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAm/MAA) nanoparticles are of potential application in targeted drug delivery. Their responsive properties in the presence of human serum albumin were investigated using dynamic light scattering (DLS), protein assay, and electron spin resonance (ESR) spectroscopy. Their interaction with human monocytes and polymorphonuclear leukocytes (PMNLs) was studied using scanning electron microscopy (SEM) and oxygen consumption method. The nanoparticles exhibited a volume phase transition at 35-40°C in Hanks balanced salt solution (HBSS) and in phosphate buffer solution (PBS) of pH 7.4. The diameter of the nanoparticles decreased slightly in the presence of HSA at 25°C at neutral pH, whereas an increase in the diameter in pH 6 PBS at 40°C was revealed. The amount of albumin adsorbed onto the nanoparticles decreased with increasing temperature. The ESR spectra of spin labeled HSA indicated a more restricted environment in the nanoparticles at elevated temperatures. The stimulation of PMNL oxygen consumption by PNIPAm based nanoparticles, an indication of phagocytosis of the particles, was not observed regardless whether the nanoparticles were incubated in plasma or serum. In contrast, the more hydrophobic polystyrene (PSt) particles induced a significant increase in the rate of oxygen consumption after the incubation. PNIPAm/MAAgrafted-PSt particles behaved similarly to the PNIPAm/MAA nanoparticles, suggesting that surface properties dictate the recognition of colloids by PMNLs.     

Significance of the extracellular bicarbonate buffer system to anaerobic glycolysis in hypoxic muscle.

1. The influence of the carbon dioxide-bicarbonate buffer system on anaerobic energy production during severe hypoxia was studied in isolated right hemidiaphragms of rats.--2. When the tissue was incubated in a Ringer solution containing 25 mM HCO-3 aerated with 7% CO2 in N2 at pH 7.4, the lactate production and lactate content of the tissue increased.--3. At an extracellular (tissue bath) pH OF 6.9 the lactate production was stimulated when carbon dioxide and bicarbonate were changed to 19% and 25 mM, respectively. This stimulatory effect disappeared when these values were lowered to 7% and 7 mM.--4. At pH 7.4 the stimulatory effect of the carbon dioxide-bicarbonate system persisted when the buffer value was lowered from 60 to 3 mM by changing the system from an open (i.e. continuous gas equilibration) to a closed one (i.e. without any gas phase). Decreasing the glucose in the media from 22 to 0 mM reduced the lactate production and abolished the stimulatory effect of the carbon dioxide--bicarbonate system.--5. There was no direct effect of this system on the glycolytic enzymes (i.e. lactate production and activity of phosphofructokinase of homogenates).     

Hypoxia in fibroblast cultures. I. Changes in glucose consumption by 5 vol.-% oxygen concentration and reduced pH.

In secondary cultures of rat embryonic fibroblasts the glucose consumption and lactate concentration were comparatively investigated in 5 and 21 Vol.-% O2 environment at pH 6.6 and pH 7.4 (HEPES-buffer). The results were correlated with cell density. The following observations were made: 1. At pH 6.6 the rate of cell proliferation was reduced to 81%; by additional hypoxia it was reduced to 71%. 2. Increase in cell density effectuated decrease of glucose consumption and lactate production at pH 7.4 and pH 6.6 in 5% as well as in 21% O2 environment. 3. At pH 7.4 enhancement of glucose consumption and lactate production due to hypoxia can be observed only at low cell density. 4. At pH 6.6 respiration of fibroblasts was little influenced by 5% O2 environment. 5. Transition from pH 7.4/21% O2 to pH 6.6/5% O2 effectuated decrease in glucose consumption and lactate concentration in tissues in hypoxic environment. This suggests a pH-governed feedback mechanism. Abbreviations used in the text: c-AMP = cyclic adenosine monophosphate; MPS = acid mucopolysaccharides (glycosaminoglycans).     

Endogenous pH shifts facilitate spreading depression by effect on NMDA receptors

Rapid extracellular alkalinizations accompany normal neuronal activity and have been implicated in the modulation of N-methyl-D-aspartate (NMDA) receptors. Particularly large alkaline transients also occur at the onset of spreading depression (SD). To test whether these endogenous pH shifts can modulate SD, the alkaline shift was amplified using benzolamide, a poorly permeant inhibitor of interstitial carbonic anhydrase. SD was evoked by microinjection of 1.2 M KCl into the CA1 stratum radiatum of rat hippocampal slices and recorded by a proximal double-barreled pH microelectrode and a distal potential electrode. In Ringer solution of pH 7.1 containing picrotoxin (but not at a bath pH of 7.4), addition of 10 microM benzolamide increased the SD alkaline shift from 0.20 +/- 0.07 to 0.38 +/- 0.17 unit pH (means +/- SE). This was correlated with a significant shortening of the latency and an increase in the conduction velocity by 26 +/- 16%. In the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), benzolamide still amplified the alkaline transient, however, its effect on the SD latency and propagation velocity was abolished. The intrinsic modulation of SD by its alkaline transient may play an important role under focal ischemic conditions by removing the proton block of NMDA receptors where interstitial acidosis would otherwise limit NMDA receptor activity.     

The effects of temperature and buffer on fibrinogen adsorption from blood plasma to glass

[125I]-Fibrinogen was used to measure the adsorption of fibrinogen from baboon plasma to two types of glass (Pyrex and a borosilicate glass) at 25 and 37 degrees C using two different buffers to dilute the plasma, the first being citrate-phosphate buffered saline (CPBSz) and the second isotonic Tris-saline (TRIS), both pH 7.4. In addition, the effects of hydration conditions, rinsing techniques, and glass-cleaning treatments on fibrinogen adsorption were evaluated. The data reveal that lower temperatures and the use of TRIS to dilute the plasma significantly enhance fibrinogen adsorption to both types of glass. As has been observed in the past, fibrinogen adsorption peaked at intermediate plasma concentrations on both Pyrex and borosilicate glass (the so-called Vroman effect), but almost twice as much fibrinogen adsorbed to glass when TRIS was used to dilute the plasma instead of CPBSz. Moreover, up to five times as much fibrinogen adsorbed to both types of glass at 25 degrees C compared with 37 degrees C. No effects of the rinsing technique or glass-cleaning treatment were observed.     

Acidic pH enhances the invasive behavior of human melanoma cells

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pH^ex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pH^ex of 7.4. This is significant, because slight changes in pH^ex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines: the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHⁱⁿ) and intracellular calcium ([Ca²⁺]ⁱⁿ), we examined the effect of these parameters on invasion. While changes in [Ca²⁺]ⁱⁿwere not consistent with invasive potential, the changes in pHⁱⁿ were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.     

Development of dengue virus plaques under serum-free overlay medium.

An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.     

A facile method to fabricate thermo- and pH-sensitive hydrogels with good mechanical performance based on poly(ethylene glycol) methyl ether methacrylate and acrylic acid as a potential drug carriers

Abstract

A thermo- and pH-sensitive hydrogel was prepared by a facile free aqueous radical copolymerization of PEGMA and AAc without any crosslinkers for controlled drug delivery. The successful fabrication of hydrogels was confirmed by Fourier transform infrared spectroscopy (FT-IR) and thermo gravimetric analysis (TGA) measurements. The morphological, mechanical and swelling properties of the obtained hydrogels were studied systematically. The results showed that the morphological and mechanical behaviors of the resultant hydrogels were strongly affected by the content of AAc. Moreover, the obtained hydrogels showed an excellent thermo-, pH- and salinity sensitivities. Release profiles of 5-Fu were studied at different pH (gastric pH 1.2 and intestinal pH 7.4) and temperatures (25?°C and 37?°C). The results showed that the release is very low at pH 1.2/37?°C and high at pH 7.4/25?°C. The cytotoxicity of hydrogels to cells was determined by an MTT assay. The result demonstrated that the blank hydrogels had negligible toxicity to cells, whereas the 5-Fu-loaded hydrogels remained high in cytotoxicity for LO2 and HepG-2 cells. Results of the present investigation exemplify the potential of this novel thermo- and pH-sensitive hydrogel for the controlled and targeted delivery of the anti cancer drug 5-Fu.