In vitro perfused-superfused cat carotid body for physiological and pharmacological studies

An in vitro perfused carotid body preparation was developed to study its chemosensory responses to physiological and pharmacological stimuli. The carotid bifurcation with the carotid body was vascularly isolated and excised from pentobarbital sodium-anesthetized cats. The CB was perfused in a chamber by gravity (80 Torr) with modified Tyrode's solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH at pH 7.40) equilibrated at a given Po2 and superfused with the same medium at (Po2 of 20 Torr). The temperature was maintained at 35.5 +/- 0.5 degrees C. The frequency of chemosensory discharges (CD) was recorded from the whole carotid sinus nerve (n = 24), and the responses were tested by repeated interruptions of perfusate flow (SF), perfusion with hypoxic medium, and injections of nicotine and cyanide (0.1 nmol to 1 mumol) and hypercapnic medium. During hyperoxic perfusion, SF resulted in a sigmoidal increase in CD, reaching a maximum that was 23.6 +/- 4.4-fold greater than the basal activity. Restoration of flow returned CD promptly to basal values. After normoxic perfusion, SF led to a similar maximal activity more rapidly, but the duration was shorter. Reduction of the perfusate PO2 (Po2 from 450 Torr to 150, 30, and less than 10 Torr) caused a nonlinear increase in CD. CO2 stimuli (PCo2 38-110 Torr) resulted in a linear increase in CD. Nicotine or cyanide increased CD in a dose-dependent manner. The preparation retained its initial responsiveness for 2-3 h, making extensive experimental studies feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro selection of RNA against kanamycin B.

Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria. In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B. After 12 cycles of selection, RNA was cloned and sequenced. Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched. Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA. It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique. The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD. The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region. A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers. The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.     

In vitro and in vivo studies with adriamycin liposomes

Liposome entrapped adriamycin retains its full cytotoxic potential when tested under $\text{in}\text{vitro}$ conditions against murine leukemia L-1210 cells. $\text{In}\text{vivo}$ drug distribution studies indicate that, relative to the free drug, a lower proportion of adriamycin administered in the liposome form is delivered to the heart and kidneys at one and four hours after injection. When administered to normal mice in high doses, anionic adriamycin liposomes appear less harmful than equal doses of the free drug as judged by alterations in animal weight gain. In these studies, a noval double packing procedure has been used for the entrapment of adriamycin in phospholipid vesicles.     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

New pharmacological studies with pentoxifylline

Polymorphonuclear (PMN) overactivation plays a critical role in microcirculation as well as in conditions such as multiorgan failure (MOF). Pentoxifylline has been shown to prevent PMN activation by endotoxin and cytokines such as TNF alpha and IL-1. In addition, MOF induced by IL-2 in animals can be prevented by pentoxifylline. The present studies evaluated two aspects of PMN activation and pentoxifylline interaction. The first was the time sequence for pentoxifylline prevention of TNF alpha activation and the second was the activity of pentoxifylline on amphotericin B activation of PMNs. TNF alpha activation of PMNs is blocked by pentoxifylline when cells are exposed to pentoxifylline prior to TNF alpha or after TNF alpha. Amphotericin B activation of PMNs was demonstrated by a decreased chemotaxis, increased chemiluminescence, and increased PMN spreading. In all conditions, pentoxifylline decreased amphotericin B activation of PMNs. These results suggest that pentoxifylline can reverse cytokine activation of PMNs and that pentoxifylline may alter some of the toxic effects of amphotericin.     

In vitro and in vivo studies with aromatase inhibitor 4-hydroxy-androstenedione

Studies with 4-hydroxyandrostenedione (4-OHA) are described which demonstrate inhibition of aromatase in human placentra and rat ovaries. In animal experiments, the compound was compared with aminoglutethimide (AG) for antitumor activity and effects on plasma hormone levels. 4-OHA was more effective than AG in causing regression of DMBA-induced hormone dependent tumors in the rat. Although estradiol concentrations in ovarian vein blood were reduced initially by both compounds, there is a reflex rise in LH and estradiol levels during long-term treatment with AG, whereas hormone levels in 4-OHA treated animals remained suppressed. Further studies in ovariectomized rats indicated that during long-term treatment, 4-OHA acts as a weak androgen (the compound has less than 1% the activity of testosterone) to directly inhibit the post-castrational rise in gonadotropin levels. This antigonadotropin action of the steroidal aromatase inhibitor may help maintain reduced ovarian estrogen secretion and thus contribute to the antitumor activity of 4-OHA.     

In vitro pharmacological properties of 4-bromodexetimide for muscarinic receptors

The decrease of m-AChR density observed hi neurodegenerative disorders has generated considerable interest in non-invasive mapping of muscarinic acetylcholine receptors (m-AChR) in the central nervous system. The ami of our study was to evaluate the selectivity of 4-bromodexetimide for the M1, M2, M3 and M4 m-AChR subtypes using in vitro binding analysis to determine the potential use of the bromine-76 labelled 4-bromodexetimide in the investigation of m-AChR subtypes in human brain with Positron Emission Tomography. Subtype selectivity of 4-bromodexetimide was determined in competition studies against tritiated subtype selective ligands using various rat or rabbit structure homogenates reflecting a single binding site and in optimal saturation and low non specific binding conditions. These conditions were reached for every subtype studied by analyzing the data from the saturation experiments of the tritiated ligands. 4-bromodexetimide displayed nanomolar affinities for the four m-AChR subtypes and a preferential selectivity for the M1 and M4 subtypes. The saturation analysis of [⁷⁶Br]4-bromodexetimide, performed with rat cortex membranes showed high affinity for m-AChR receptors (K_d = 1.8 nM). As in vivo studies of [⁷⁶Br]4-bromodexetimide showed preferential localization in the cortex and the striatum which are M1 and M4 rich structures and since it binds preferentially to the M₁ and M₄ subtypes, this radiotracer can still allow a combined subtype specific measurement of these muscarinic receptors.     

In vitro and in vivo mutagenicity studies with airborne particulate extracts

The contribution of nitro compounds to airborne particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8DNP6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 mg/kg either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 mg/kg (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.     

In vitro studies of histone glycation

Reductive amination of histone H1 by [U-14C]glucose was performed in the presence of sodium cyanoborohydride and was approximately proportional to the glucose concentration. Lysine was the principal amino acid substituted. Glycation also occurred in the absence of cyanoborohydride. Browning reactions of histones were monitored by delta A 325 whereby it was shown that glucose 6-phosphate was more reactive than glucose and that each of the histone fractions reacted with glucose 6-phosphate giving the browning reaction.     

In vitro studies of iron bioavailability

The bioavailability of iron from foods is ultimately determined by interactions between iron and other components in the digestive milieu. Perhaps the most important factor is the concentration of Fe2+ during transit through the duodenum. During in vitro simulations of human digestion it is possible to probe the concentration of Fe2+, the rate of Fe2+ formation, and total iron concentration using ferrous chromogens. It is crucial, of course, that the chromogen not interfere with the redox reactions occurring during digestion. Accordingly, ferrozine was examined with regard to its ability to reduce complexes Fe3+, alter rates of Fe3+ production, detect Fe2+ present in the digestive mixture and differentiate the effects of chelating and reducing agents in the mobilization of iron from pinto beans. The chromogen was found to be free from apparent artefacts and to be a sensitive and reproducible probe of the state of iron in digestive mixtures.     

In vitro studies of mycoplasma-like organisms

We determined whether the western X mycoplasma (WXM) isolated from Colladonus montanus could be maintained in vitro by ultrathin sections or by assay of infectivity. Large spherical or small electron-dense bodies like those found in intact infected cells were observed in some media. Infectivity of WXM can be maintained for 28 days in cultured salivary glands in a newly developed medium, and for 281 days (seven passages) in modified AcTc, for 231 days (eight passages) in modified PC, 107 days (one passage) in spiroplasma medium, and 52 days (one passage) in modified GITC medium extracts. However, there is no evidence that WXM multiplied in any medium.