Nonpenetrating Agents Research Articles

Research and application of anti - corrosion cement slurry in formation water corrosion environment

Corrosion of cementing cement ring causes the cement ring permeability to increase, resulting in serious casing corrosion. The casing corrosion perforation leads to an increase in water content of oil wells, low driving use of water injection wells, and imbalance of injection and production well patterns. Therefore, on the basis of conventional cementing cement, epoxy resin emulsion non-penetrating agent and corrosion inhibitor composed of rust and antibacterial materials are added to reduce the corrosion and permeability of cementing cement and reduce casing corrosion. The cement sample permeability experiment shows that adding 10% non-permeability agent and 2% corrosion inhibitor to low-density cement reduces the cement stone sample permeability from 0.075mD to 0.021mD, a decrease of 3.57 times. In the conventional density cement, adding 10% non-penetrating agent and 2% corrosion inhibitor, the permeability of the cement stone sample decreased from 0.041mD to 0.0009mD, a decrease of 4.56 times. The evaluation experiment of cement stone anti-corrosion performance shows that adding 10% non-penetrating agent and 2% corrosion inhibitor to cement can increase the corrosion resistance of cement samples by 6 times. At the same time, the thickening time and compressive strength of cement stone samples remain unchanged compared with conventional cement. Optimizing the technological measures of supporting anti-corrosion tools, and summing up a set of anti-corrosion cementing technology through the field application of anti-corrosion cement slurry, which provides strong technical support for the development of cementing technology under the corrosive environment of formation water in the oil field.

Impact of Short-Term Combined Antiretroviral Therapy on Brain Virus Burden in Simian Immunodeficiency Virus-Infected and CD8 + Lymphocyte-Depleted Rhesus Macaques

Antiretroviral drugs suppress virus burden in the cerebrospinal fluid of HIV-infected individuals; however, the direct effect of antiretrovirals on virus replication in brain parenchyma is poorly understood. We investigated the effect of short-term combined antiretroviral therapy (CART) on brain virus burden in rhesus monkeys using the CD8-depletion model of accelerated simian immunodeficiency virus (SIV) encephalitis. Four monkeys received CART (consisting of the nonpenetrating agents PMPA and RCV) for four weeks, beginning 28 days after SIV inoculation. Lower virus burdens were measured by real-time RT-PCR in four of four regions of brain from monkeys that received CART as compared with four SIV-infected, untreated controls; however, the difference was only significant for the frontal cortex (P < 0.05). In contrast, significantly lower virus burdens were measured in plasma and four of five lymphoid compartments from animals that received CART. Surprisingly, despite normalization of neuronal function in treated animals, the numbers of activated macrophages/microglia and the magnitude of TNF-alpha mRNA expression in brain were similar between treated animals and controls. These results suggest that short-term therapy with antiretrovirals that fail to penetrate the blood-cerebrospinal fluid barrier can reduce brain virus burden provided systemic virus burden is suppressed; however, longer treatment may be required to completely resolve encephalitic lesions and microglial activation, which may reflect the longer half-life of the principal target cells of HIV/SIV in the brain (macrophages) versus lymphoid tissues (T lymphocytes).

Theoretical considerations for oocyte cryopreservation by freezing

Attempts to cryopreserve oocytes by freezing have, to date, been based mostly on empirical approaches rather than on basic principles, and perhaps in part for this reason have not been very successful. Theoretical considerations suggest some fairly ‘heretical’ conclusions. The concentrations of permeating cryoprotectants employed in past studies have probably been inadequate, and the choice of propylene glycol (PG) as a protective agent is questionable. The use of non-penetrating agents, such as sucrose to preshrink oocytes prior to freezing and which, therefore, exacerbate osmotic stress during freezing, may be inappropriate, yet may protect in part by reducing the concentration of PG during freezing. The methods used to add and remove cryoprotectant may be suboptimal, and may be based on an inadequate understanding of the cryobiological constraints for oocyte survival. Given these concerns, it is not surprising that fully satisfactory results have been elusive, but there is every reason to believe that greater success is possible using a more theoretically appropriate approach.

Effect of Selection of Molecular Descriptors on the Prediction of Blood−Brain Barrier Penetrating and Nonpenetrating Agents by Statistical Learning Methods

The ability or inability of a drug to penetrate into the brain is a key consideration in drug design. Drugs for treating central nervous system (CNS) disorders need to be able to penetrate the blood-brain barrier (BBB). BBB nonpenetration is desirable for non-CNS-targeting drugs to minimize potential CNS-related side effects. Computational methods have been employed for the prediction of BBB-penetrating (BBB+) and -nonpenetrating (BBB-) agents at impressive accuracies of 75-92% and 60-80%, respectively. However, the majority of these studies give a substantially lower BBB- accuracy, and thus overall accuracy, than the BBB+ accuracy. This work examined whether proper selection of molecular descriptors can improve both the BBB- and the overall accuracies of statistical learning methods. The methods tested include logistic regression, linear discriminate analysis, k nearest neighbor, C4.5 decision tree, probabilistic neural network, and support vector machine. Molecular descriptors were selected by using a feature selection method, recursive feature elimination (RFE). Results by using 415 BBB+ and BBB- agents show that RFE substantially improves both the BBB- and the overall accuracy for all of the methods studied. This suggests that statistical learning methods combined with proper feature selection is potentially useful for facilitating a more balanced and improved prediction of BBB+ and BBB- agents.

COS-7 cells stably transfected to express the human ETB receptor provide a useful screen for endothelin receptor antagonists.

Endothelin acts via specific membrane-bound receptors through signal transduction pathways that include increases in intracellular free calcium and inositol triphosphate generation. Two endothelin receptors have been cloned. The ETA receptor is ET-1 selective, and the ETB receptor is isopeptide nonselective. Both receptor subtypes are widely distributed throughout the body, although ETA receptors predominate in vascular smooth muscle, whereas ETB receptors predominate in the brain. The presence of mixed receptor subtypes makes functional screening of subtype-specific analogues difficult. A eukaryotic expression vector was constructed by inserting the cloned coding region of the human ETB receptor downstream from the Rous sarcoma promoter. COS-7 cells were transfected with this construct, and cell lines were isolated with stably integrated copies of the relevant gene. One line, 1C7, was shown to specifically bind 125I-ET-1. Scatchard analysis indicated a Kd value of 8.8 pM and a Bmax value of 1.02 pM/mg. ET-1 stimulated phosphoinositide hydrolysis in a dose-dependent manner, as did ET-3, sarafotoxin 6c, and [1,3,13,15Ala]ET-1, whereas BQ123, a selective ETA receptor antagonist, did not inhibit the action of ET-1. The transfected receptor stimulates phosphoinositide (PI) hydrolysis via a pertussis-sensitive pathway. Pretreatment of the membrane from 1C7 cells with dithio-bis-nitrobenzoic acid (DTNB) a negatively charged, nonpenetrating agent capable of oxidizing sulfhydryl groups, and N-ethyl-maleimide (NEM), a penetrating agent that causes irreversible alkylation of sulfhydryl groups, significantly reduces Bmax but has no effect on Kd. In whole cells, DTNB pretreatment abolishes the ability of ET-1 to stimulate PI hydrolysis.

Characterization and Control of Expression of Cell Surface Aminopeptidase N Activity in Human Mesangial Glomerular Cells

Intact human glomerular mesangial cells in culture hydrolyzed synthetic substrates of aminopeptidase N. Mesangial cell aminopeptidase N is an ectoenzyme as demonstrated by the extracellular location of the product, the inhibition of enzyme activity by the diazonium salt of sulfanilic acid, a nonpenetrating agent, the similar activities measured with intact and sonicated cells and the localization of the immunoreactive antigen by immunofluorescence on the cell surface. A broad substrate specificity was documented with various <i>p</i>-nitroanilide substrates in the following order of affinity: Ala, Leu > Lys, Arg > Pro, Gly and Val. Using Ala <i>p</i>-nitroanilide as substrate, apparent K_m and V_max values of 0.86 ± 0.70 m<i>M</i> and 3.54 ± 0.13 nmol min^–1 mg^–1, respectively were obtained. Enzyme activity was inhibited by bestatin and 1, 10-phenanthroline. Inhibition by 1 10-phenanthroline was noncompetitive. Cell surface aminopeptidase N activity increased in the presence of phorbolmyristate acetate (PMA). This effect was time-dependent, requiring a lag time of 12 h. It was also concentration-dependent with a threshold at 1 ng/ml and a maximum stimulation (2.2 times basal value) at 10 ng/ml. The effect of PMA was prevented by cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthesis. It was also suppressed by H7, an inhibitor of protein kinase C activity. The presence of aminopeptidase N in cultured glomerular mesangial cells and control of its expression by mitogens suggest that this enzyme may play a role in glomerular cell proliferation.

Interaction between advancing ice fronts and erythrocytes

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) are excluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells are engulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated.

Phagocytosis-induced chemotaxis receptor cycling in neutrophils is mediated by thiol oxidation

We previously demonstrated that neutrophil (PMN) phagocytosis of opsonized zymosan (OPZ) caused oxygen-dependent inhibition of chemotactic peptide receptor (CPR) binding using the ligand 3H-formyl- methionyl-leucyl-phenylalanine (3H-FMLP). In the current studies we sought to determine the mechanism of CPR inhibition by OPZ. We found that 3 mM cysteine and 5 mM dithiothreitol (DTT) did not decrease PMN phagocytosis, but abolished CPR inhibition by OPZ. Furthermore, incubation of PMN with DTT after OPZ partially restored the suppressed CPR. PMN CPR saturation studies with 3H-FMLP on PMN incubated with or without DTT after phagocytosis indicated that DTT restored receptor numbers to 92% +/- 6% of prephagocytosis values and also improved CPR affinity for 3H-FMLP. In additional studies we found that the cell penetrating thiol reagents mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and diamide, but not the nonpenetrating agents p- chloromercuriphenylsulfonic acid and p-chloromercuribenzoate, caused rapid, dose-dependent, DTT-inhibitable suppression of up to 85% CPR binding. CPR inhibition by HgCl2 and NEM was irreversible. PMN CPR saturation studies showed that NEM decreased total receptor numbers, not CPR affinity. The effect of NEM was not inhibited by receptor occupancy at 4 degree C or at 37 degree C. These studies indicate that: (1) intact thiol groups are a requirement for CPR ligand-binding; (2) the thiol groups are presumptively located on the interior side of, or within hydrophobic portions of, the plasma membrane and are not part of the 3H-FMLP binding site; (3) thiol oxidation-reduction induces reversible alteration in CPR binding; (4) phagocytosis-induced modulation of the CPR is due, in part, to thiol oxidation. These studies suggest that postphagocytic, thiol-mediated reduction in CPR binding may play a physiologic role in the inhibition of PMN chemotaxis at inflammatory sites. The state of CPR-associated thiols may be an important determinant of CPR binding under physiologic conditions other than phagocytosis.

Phagocytosis-Induced Chemotaxis Receptor Cycling in Neutrophils Is Mediated by Thiol Oxidation

We previously demonstrated that neutrophil (PMN) phagocytosis of opsonized zymosan (OPZ) caused oxygen-dependent inhibition of chemotactic peptide receptor (CPR) binding using the ligand 3H-formyl-methionyl-leucyl-phenylalanine (3H-FMLP). In the current studies we sought to determine the mechanism of CPR inhibition by OPZ. We found that 3 mM cysteine and 5 mM dithiothreitol (DTT) did not decrease PMN phagocytosis, but abolished CPR inhibition by OPZ. Furthermore, incubation of PMN with DTT after OPZ partially restored the suppressed CPR. PMN CPR saturation studies with 3H-FMLP on PMN incubated with or without DTT after phagocytosis indicated that DTT restored receptor numbers to 92% +/- 6% of prephagocytosis values and also improved CPR affinity for 3H-FMLP. In additional studies we found that the cell penetrating thiol reagents mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and diamide, but not the nonpenetrating agents p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate, caused rapid, dose-dependent, DTT-inhibitable suppression of up to 85% CPR binding. CPR inhibition by HgCl2 and NEM was irreversible. PMN CPR saturation studies showed that NEM decreased total receptor numbers, not CPR affinity. The effect of NEM was not inhibited by receptor occupancy at 4 degree C or at 37 degree C. These studies indicate that: (1) intact thiol groups are a requirement for CPR ligand-binding; (2) the thiol groups are presumptively located on the interior side of, or within hydrophobic portions of, the plasma membrane and are not part of the 3H-FMLP binding site; (3) thiol oxidation-reduction induces reversible alteration in CPR binding; (4) phagocytosis-induced modulation of the CPR is due, in part, to thiol oxidation. These studies suggest that postphagocytic, thiol-mediated reduction in CPR binding may play a physiologic role in the inhibition of PMN chemotaxis at inflammatory sites. The state of CPR-associated thiols may be an important determinant of CPR binding under physiologic conditions other than phagocytosis.

Optimal temperature ranges for control of cooling rate

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between −5 and −20 °C followed by rapid cooling to −196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to −30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at −196 °C.

Differing actions of penetrating and nonpenetrating cryoprotective agents

A two-step freezing technique has been used to examine the role of cryoprotective agents during cooling. Chinese hamster fibroblasts were cooled to various subzero holding temperatures and subsequently thawed or cooled to −196 °C before thawing. Cells were suspended in various concentrations of dimethylsulfoxide (DMSO) or hydroxyethyl starch (HES) before freezing. The results indicated differing protective actions of DMSO and HES. These differences were verified using glycerol as either a penetrating or a nonpenetrating agent. The results are consistent with the concepts that cryoprotection is based on the avoidance or minimization of intracellular freezing and the minimization of damage to the cell from the environment of concentrated solutes during cooling, and that the colligative action of both penetrating and nonpenetrating agents allows the cells to survive the conditions for a reduction of cell water content during cooling thereby reducing the amount of intracellular freezing. The results indicate that penetrating and nonpenetrating agents accomplish this in different ways. Penetrating agents create the environment for a reduction of cell water content at temperatures sufficiently low to reduce the damaging effect of the concentrated solutes on the cells. Nonpenetrating agents osmotically “squeeze” water from the cells primarily during the initial phases of freezing at temperatures between −10 and −20 °C when these additives become concentrated in the extracellular regions.

Differing cryoprotective actions of penetrating and nonpenetrating agents with tissue culture cells

Differing cryoprotective actions of penetrating and nonpenetrating agents with tissue culture cells

Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to −196 °C

Washed human erythrocytes were cooled at different rates to −196 °C in the presence of different concentrations of both penetrating and nonpenetrating cryoprotective additives, and thawed rapidly. The protective effect of albumin when ACD blood is frozen was demonstrated. All compounds tested (glycerol, dimethylsulfoxide, sucrose, bovine serum albumin and polyvinylpyrrolidone) protected the red cells at freezing rates less than that giving optimal survival, and with the exception of polyvinylpyrrolidone, all compounds in high concentration increased freezing damage at faster cooling rates. The net result was to shift the cooling rate giving optimal survival to slower values. The increase in damage at faster rates in high concentrations of the nonpenetrating agents sucrose and bovine serum albumin was unexpected and casts some doubt on the idea that under these conditions nonpenetrating compounds protect the cells by withdrawing water that could otherwise form intracellular ice.

Cryoprotective agents

Two forms of artificial cryoprotection are immediately available for experimental use. Foremost are the penetrating agents functioning on a colligative basis and used in concentrations of 2–4 m. These apparently function simply by preventing the concentration of extracellular solution and thus reducing cell dehydration to a tolerable degree. There are two immediate obstacles to the general use of such compounds. First, the penetration of cells must be uniform and achieved without the imposition of osmotic stresses which can in themselves be destructive. The particular virtues of DMSO, ethanol, and methanol are the speed and apparent universality of their penetration. The major defect of glycerol is its slow movement across those membranes which are permeable to it and the largo number of tissues which appear to be virtually impermeable. The second obstacle is the toxicity of penetrating agents in the high concentrations necessary to achieve protection. Ethanol and methanol can be ruled out on this basis, and studies of DMSO, dimethylformamide, and ethylene glycol have not been particularly encouraging. A second means of cryoprotection is through the use of nonpenetrating agents. We have presented evidence which suggests that the efficacy of nonpenetrating cryoprotective agents lies in their ability to enable the cell membrane to leak solute reversibly under osmotic stress. The success of such an approach presumably depends upon the achievement of a freezing rate which is rapid enough to limit solute leak to low-molecular-weight solutes, prevent loss of the larger and more essential protoplasmic elements and yet not be so fast as to produce intracellular freezing. It will also be essential that the cell have an opportunity to heal and restore its normal solute content. This hypothesis for the mechanism of extracellular cryoprotection also provides an explanation for the general failure of extracellular agents to afford significant cryoprotection for packed cells, tissues, or organs. If the cell avoids excessive dehydration through the influx of extracellular solute, it is clearly necessary that a substantial amount of extracellular solute be available which is only the case in a relatively dilute cell suspension. In a tissue, extracellular solute is present in such insignificant amounts as to be of little value in facilitating the leak mechanism. The provision of a suitable environment for cell healing ex vivo is also difficult to achieve. Ideally, the prevention of nucleation would provide the simplest and most effective means of avoiding freezing injury but whether compounds which will do this exist and whether they could be artificially introduced into animal cells can at the moment be no more than speculation. The most promising approach to the preservation of organized tissues and organs through artificial cryoprotection would still appear to be through the development of nontoxic, rapidly penetrating cryoprotective agents or means of preventing their toxicity or of reversing its effects.