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Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules.

The in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.

Animals↗

Measurement of edema in the nervous system. Use of Percoll density gradients for determination of specific gravity in cerebral cortex and white matter under normal conditions and in experimental cytotoxic brain edema.

A method is presented by which density measurements can be performed on samples from cerebral cortex and white matter of normal and intoxicated animals using nontoxic ingredients as an alternative to the bromobenzene-kerosene technique described by Nelson et al. (1971). A continuous density gradient is prepared in a calibrated glass cylinder by using a new product, Percoll, which consists of colloidal silica particles coated with polyvinyl pyrrolidone. The gradient is stable and the same column can be used for repeated experiments over a long period of time. Interactions between the gradient media and the samples are evaluated and various methodological aspects concerning removal and handling of the tissue samples are presented. Experiments with acute triethyltin (TET) intoxication in the mouse and the hamster show that the Percoll technique can be used as an alternative to the bromobenzene-kerosene method in quantitative studies on cytotoxic brain edema.

Animals↗

An improved Percoll density gradient for measurements of experimental brain edema. Addition of sucrose to an isotonic gradient in an attempt to balance osmotic conditions during density determinations.

Microgravimetric methods are very useful for quantitative studies on brain edema. One of the techniques available is based on a gradient made up by NaCl and polyvinyl pyrrolidone-coated silica particles (Percoll). The present study was performed to find a way of minimizing fluid shifts between the gradient and the samples. For this purpose, five Percoll density gradients containing various concentrations of sucrose in isotonic saline were prepared. Equivalent samples of normal mouse brain were then added and their second slow movement (drift) indicating interactions between the tissue and the gradient was followed. A concentration of 0.125 M sucrose eliminated the drift of the samples almost entirely. The capacity of this sucrose-containing gradient to reveal brain edema was then evaluated by comparing the density values obtained with those measured in the traditional bromobenzene-kerosene gradient as described by Nelson et al. (1971). For this purpose, we produced in the mouse an acute cytotoxic edema by triethyltin intoxication and a vasogenic edema by a cortical cryogenic injury. The two gradients showed almost identical results. We conclude, therefore, that the 0.125 M sucrose-containing Percoll gradient is a very good alternative to bromobenzene-kerosene gradients used for brain density determinations. Furthermore, Percoll gradients are very stable and contain only non-toxic ingredients.

Animals↗

Evaluation of polyvinyl alcohol hydrogel as a soft contact lens material.

We prepared a transparent polyvinyl alcohol (PVA) hydrogel from a PVA solution in a mixed solvent consisting of water and a water-miscible organic solvent by cooling. The physical properties of the hydrogel were evaluated in various mixed solvents and compared with those of commercially available soft contact lens materials, such as polyhydroxyethyl methacrylate (PHEMA) and copolymers of methylmethycrylate (MMA) and N-vinyl pyrrolidone (N-VP). The PVA hydrogel showed higher tensile strength and elongation before breaking than did the other materials. Also, the PVA hydrogel was comparable in its high water content and its oxygen permeability with the MMA/VP copolymers. The protein adsorption of the PVA hydrogel was much lower than that of the other materials. Soft contact lenses of PVA hydrogel were applied to rabbit eyes for 12 weeks. The effects of the lenses on the cornea were studied by biomicroscopy, ultrasonic pachymetry, and histopathologic examination. No abnormal findings were noted, suggesting that the PVA hydrogel may be promising as a new material for use in soft contact lenses.

Animals↗

Electron microscopic demonstration of glycogen and phosphorylase activity in rat hepatocytes.

The effect of fixation with a bicarbonate-buffered solution of paraformaldehyde and polyvinyl pyrrolidone (PVP) on the ultrastructural demonstration of glycogen and phosphorylase activity in rat hepatocytes has been studied. Phosphorylase was demonstrated by the precipitation of liberated phosphate ions with ferrous ions. 7.5% PVP was included in all steps in the procedure before post-fixaiton in osmium tetroxide. Glycogen particles were well preserved. Structures connecting membranes and glycogen particles were also evident. Phosphorylase activity was rapidly inhibited by the fixative; the fixation time was, therefore, kept very short. The final reaction product was localized on glycogen particles and on endoplasmic membranes in association with glycogen particles. The results support the view that endoplasmic membranes are involved in the metabolism of glycogen in hepatocytes.

Animals↗

The use of hyaluronidase and glucosidase to remove mucus from the rat middle ear cavities for SEM studies.

To conduct SEM studies on epithelium containing mucus-producing cells it is essential to remove the mucus which normally obscures the epithelial surface. This study presents a method which effectively removes the covering layer of mucus in the rat middle ear. Healthy Sprague-Dawley rats were decapitated and the middle ears dissected free. Incubation and agitation of the middle ear specimens in hyaluronidase (50 IE ml-1) and/or glucosidase (8%) removed the mucus from the middle ear cavity without altering the surface structures. It was also revealed that substances such as polyvinyl-pyrrolidone (PVP) (used to increase the colloid osmotic pressure of, e.g., the fixative solution) must be omitted when preparing ciliated specimens for SEM.

Animals↗

The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells.

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low Km cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH4C1) cells in culture (ED50 = 5.10(-8) M). This effect is due to a selective inhibition of the low Km cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca2+/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the alpha i family (G alpha i2 or Gk). Rolipram did not affect phosphoinositide metabolism (i.e. IP3 accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20-1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity. Long term incubation of GH4C1 cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of beta-receptor number, AC activation and cAMP accumulation, while Ro 20-1724 brought about a marked down-regulation and desensitization of the AC complex. In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in beta-adrenoceptor AC downregulation. These features are not shared by the other drugs tested.

3',5'-Cyclic-AMP Phosphodiesterases↗

Width and lattice spacing in radially compressed frog skinned muscle fibres at various pH values, magnesium ion concentrations and ionic strengths.

The width (D) and the 1,0 lattice spacing (d1,0) at various ionic compositions of mechanically skinned single fibres (from semitendinosus muscle of Rana catesbeiana) were measured at various concentrations of polyvinyl pyrrolidone (PVP K-30, Mn = 40 000) from 0 to 6% at 20 degrees C. In a standard relaxing solution (4 mM MgATP2-, 1 mM Mg2+, 4 mM EGTA, ionic strength 150 mM and pH 7), d1,0 decreased exponentially as the PVP concentration increased: d1,0 was 41.3 +/- 0.4 (mean +/- S.D.) nm at 0% PVP and 32.9 +/- 0.4 nm at 6% PVP. D was proportional to d1,0 except at very low PVP concentrations, i.e. at 1% PVP, D decreased by 7%, whereas d1,0 decreased by only 3%. At 0% PVP, D and d1,0 decreased when either pH or ionic strength (gamma/2) was lowered. At 6% PVP, D and d1,0 decreased with lowered pH or increased [Mg2+], but was independent of gamma/2. The radial stiffness, or degree of resistance to the changes of D against the compressing force, increased considerably at d1,0 less than or equal to 35 nm in a standard relaxing solution, but not at pH 5.5 or 30 mM [Mg2+]. These effects of pH, [Mg2+] and gamma/2 on D or d1,0 and on the radial stiffness can be explained by the modification of the properties of the elastic element and the hinge between subfragment-1 and -2 and/or the hinge between subfragment-2 and light meromyosin.

Animals↗

Effects of cigarette smoke on the antibody responses to thymic independent antigens from different lymphoid tissues of mice.

The influence of cigarette smoke on the humoral immune response of mice was investigated in lymphocytes derived from the spleen, bone marrow (BM) and mesenteric lymph nodes (MLN). Mice of the DBA/2J or C57BL/6 strain were exposed to cigarette smoke of a standard research cigarette, 2R1, twice a day, ten puffs each in morning and afternoon for 20, 40 or 60 weeks. At the end of the smoking period, animals were immunized intraperitoneally with the thymic independent antigens polyvinyl pyrrolidone (PVP) or trinitrophenyl (TNP)-Ficoll. The antibody responses were analyzed using sheep red blood cells coated with PVP or TNP, in a plaque forming cell (PFC) assay. The results indicate a statistically significant inhibition of the antibody response induced by PVP but not by TNP-Ficoll in splenic B cells of smoke exposed mice compared to sham controls. When tested in other lymphoid organs, there was higher anti-TNP PFC response from the BM and MLN cells of smoke exposed animals compared to sham controls.

Animals↗

Is glutamic acid the pyramidal tract neurotransmitter?

Applied by microiontophoresis, 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) antagonized excitation by glutamic acid but not by acetylcholine of neurones in the rat cuneate nucleus. HA-966 blocked the short latency excitation of cuneate neurones following stimulation of the pyramidal tract on 28 of 40 cells (70%). Thus, glutamate or a related amino-acid may be the neurotransmitter released by pyramidal tract neurones.

Acetylcholine↗

Prevalence of Gemella haemolysans on the pharyngeal mucosa of man.

By culturing pharyngeal swabs from 199 students a carrier rate for Gemella haemolysans of 29.7% was detected. Demonstration of hemolysis depended on blood species and on agar base. Optimum growth was obtained under aerobic conditions in a 10% CO2-enriched atmosphere. The cells divided in two planes which were not regularly at right angles to each other. They appeared to be surrounded by a small capsule. Contrary to earlier descriptions, acid was produced from galactose, acetoin was produced by practically all strains and nitrite was reduced by all strains. Acid production from trehalose and N-acetyl-glucosamine, alkaline and acid phosphatase, C4 and C8 esterase, pyrrolidone arylamidase and phosphoamidase activities were detected. In tube precipitation, antigenic relations between type strain and the new isolates were demonstrated.

Adult↗

Measurement of active constitutive L-pyrrolidonyl-peptidase from the genera Streptococcus and Enterococcus.

In the family Streptococceae the ability to measure L-pyrrolidonyl-peptidase is limited to Lancefield group D Enterococcus and group A Streptococcus pyogenes. A number of methods exist to assay this enzyme. All measure pyrrolidonyl-peptidase by the ability of the bacterium to cleave L-pyrrolidonyl-beta-naphthylamide to form free beta-naphthylamine and L-pyrrolidone-carboxylic acid. Free beta-naphthylamine is then reacted with N,N-dimethylamino-cinnamaldehyde to form a red color complex. These methods are generally expensive and require a 2-4 h incubation period before results are available. A method, which employs the substrate dried on paper discs and can be easily made, is described herein. It is simple to perform, inexpensive, rapid, and has a long storage life. The results of this constitutive method are equivalent to those obtained using a commercial system.

Aminopeptidases↗

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: delayed emergence of the memory retention effects.

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10-12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10-12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [3H]SCH 23390 or [3H]spiperone binding from D1 or D2 dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.

Animals↗

Influence of accompanying anion on intestinal radiocalcium absorption.

To assess directly the effect of ionic dissociation on the bioavailability of calcium, we used the double isotope inverse convolution method to compare the absorption of calcium gluconate and calcium pyrrolidone carboxylate, an organic, highly dissociated salt. Two tests were performed at a 2 day interval, using in random sequence either salt as a carrier. Forty-eight subjects of various age and clinical condition were studied. The use of the more dissociated salt consistently and significantly increased fractional absorption in a rather constant ratio. Moreover, it slowed absorption in normal subjects whatever their age, and accelerated it in patients with chronic renal failure or osteoporosis, leading to inferences on the alteration of calcium absorption in these conditions.

Adult↗

Cell proliferation on hydrogels.

The adhesion and proliferation of mammalian fibroblasts (Flow 7000) on the surface of hydrophilic (copolymer of N-vinyl-2-pyrrolidone and methyl methacrylate) and hydrophobic [polymethylmethacrylate (PMMA) stereocomplex] hydrogels with a wide range in water content were studied morphologically and quantitatively. It was demonstrated that cell proliferation on hydrogels by a static culture method decreased as the water content of the gels increased. However, it is remarkable that the cell proliferation on PMMA hydrogels with a high water content is equivalent to that on glass Petri dishes. The results obtained in the proliferation of cells on the surface of these hydrogels closely correspond to the state of cell adhesion. When fresh medium or air was perfused from the opposite side of the PMMA hydrogel membrane on which the cells were proliferating (perfusion method), the cells continued to grow into a higher density than with the conventional static culture method. In the case of fresh medium perfusion, the amount of proliferated cell was dependent on both the permeability of the membrane and the density of the membrane "scaffolding." Virus multiplication in the cultured cells increased in proportion to the cell density, whereas the cell function was similar in both culture methods.

Cell Adhesion↗

Isolation of Trypanosoma cruzi from blood by histopaque and continuous percoll gradient centrifugations.

Separation of the blood forms of trypanosomes from the blood of infected animals is difficult, especially in the case of Trypanosoma cruzi Y strain. Two procedures to isolate the Y strain blood forms of T. cruzi using polyvinyl pyrrolidone-coated silica (percoll) and histopaque are reported in this study. The recovery rates of parasites were 16 +/- 5 and 68 +/- 16%, respectively. The parasites isolated by these methods presented normal motility and morphology and were infective to albino mice with prepatent periods, parasitemia curves, and polymorphism patterns during the infection that were similar to those of control parasites. In addition, the preservation of surface antigens was confirmed by immunocytochemical studies.

Animals↗

Blood substitutes.

Substitutes for whole blood include blood fractions such as plasma, serum albumin and other fluids of various kinds which are not derived from blood but are used as plasma volume expanders; these, include the usual crystaloid intravenous solutions. Since in comparison to blood far more of these later solutions are given intravenously, a thorough knowledge of plasma volume expanders is essential. The first use of such expanders in human patients was by Hogan in 1915. He used colloidal gelatin and noted an improvement in blood pressure in shock. In 1945, Gronwall and Ingelman advocated the use of dextran in shock. The reguirements for an acceptable plasma substitute are: a satisfactory colloidal osmotic pressure, constand composition at reasonable cost, a viscosity suitable for intravenous administration, stability in prolonged storage at variable temperatures, and sterilization by autoclaving. Such substances must be either fully excreted or metabolized, and must cause no early or late tissue damage. They must be non-antigenic and pyrogen free. They must cause no change in the blood such as haemolysis, R.B.C. agglutination, increased sedimentation rate and no impairment of haemostasis. The presently available plasma expanders include blood derivatives (plasma, albumin), modified protein (gelatin, oxypolygelatin), polymerized carbohydrates (dextran) and plastics (polyvinyl pyrrolidone-PVP). All these substances expand plasma volume, decrease haematocrit and plasma proteins, increase sedimentation rate and blood pressure. Dextran, PVP and geletin do not alter hepatic function. Dextran and gelatin have no deleterious effects on renal function. Features of the clinically used plasma expanders are: 1. Fresh Frozen Plasma Fresh frozen plasma contains all clotting factors except platelets. The risk of the transmission of hepatitis is present as it is with whole blood. 2. Plasma Protein Fractions Plasma protein fractions are free of hepatitis virus, but may cause arteriolar dilatation and hypotension. 3. Serum Albumin Serum albumin is a concentrated blood protein fraction. It is salt poor, stable and does not transmit the virus of hepatitis. Since it has a high oncotic pressure it is necessary to give significant quantities of clear fluids with it. It is expensive, scarce, and dilutes the clotting factors. It is, however, a first choice for emergency treatment of shock; 4. Dextran The dextrans may be of medium or low molecular weight. They are inexpensive and readily available, and do not transmit the virus of hepatitis. In large amounts they cause a coagulation defect and may be antigenic. Continued.

Blood Proteins↗