Comparison of the effects of CRF and stress on levels of pituitary cyclic AMP and plasma ACTH in vivo

We have previously reported that acute stress increases levels of rat pituitary cyclic AMP in vivo. The present study was conducted to test the hypothesis that stress-induced increases in pituitary cyclic AMP in vivo were mediated via CRF. We compared the effects of various stressors with the effects of CRF or epinephrine administration on pituitary cyclic AMP and plasma ACTH responses in vivo. Stressors, epinephrine or CRF increased levels of pituitary cyclic AMP. Pituitary cyclic AMP response to either immobilization or CRF was much greater at light onset than at lights off in rats maintained on at 12 hr light: 12 hr dark lighting regimen. In rats with pituitary stalk transections, footshock did not increase levels of pituitary cyclic AMP, suggesting that some factor of central origin was required for this stress response. Exogenous CRF administration did increase levels of pituitary cyclic AMP in stalk-transected rats, while epinephrine increased levels in sham-operated but not in stalk-transected rats. Antisera to CRF markedly decreased pituitary cyclic AMP response to exogenous CRF administered 6 min following antisera and partially attenuated pituitary cyclic AMP response to forced running. Taken as a whole these data support a major role for CRF in the pituitary cyclic AMP response to stress.     

Flavonoids protect against oxidative damage to LDL in vitro: use in selection of a flavonoid rich diet and relevance to LDL oxidation resistance ex vivo?

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 ± 1.6 min compared to 49 ± 2.1 min).     

Chronotoxicity of a Copper (II) Complex with a Linear Tetradentate Ligand in Mice

In vitro copper (II) complex presents antimitotic effects. In this work, we have studied the in vivo seasonal toxic effects of copper (II), ligand (H₂L) and the complex [Cu(H₂L)(H₂O)₂]Cl₂·4H₂O in male Swiss mice. During spring, an i.p. injection of CuCl₂ in aqueous NaCl (9 g·l^-1) up to 0.05 µmol·kg^-1 b.w. (body weight) killed 60% of the rodents after 6 days. LD100 was up to 0.3 µmol·kg^-1; H₂L was well tolerated, while the complex was 30% lethal with 50 µmol·kg^-1. In autumn, mice were less sensitive to CuCl₂, and both ligand and complex were equally tolerated and this leads to the conclusion that, in vivo, chronotoxicities of copper (II) and complex in NaCl aqueous solutions are quite different in spring and autumn seasons.     

A study of chlorophyll b in vivo

1. The absorption spectrum of chlorophyll b in vivo at 77°K is presented as the difference spectrum between preparations of spinach and chlorophyll b-free Vischeria stellata chloroplasts.

2. A shoulder on this spectrum around 662 nm is due to a component different from chlorophyll b. This component may well be identical with the chlorophyll a form, chlorophyll a (665).

3. The 77°K chlorophyll b absorption spectra in the nonfractionated photosyn-thetic pigment apparatus and in fractions mainly representing Photosystems 1 or 2 are not significantly different.

4. The aerobic irreversible photobleaching of chlorophyll b was studied in the intact pigment complex as well as in fractions mainly consisting of Photosystem 1 or 2. A two-step photobleaching was observed in all cases. The time-course of this bleaching was not significantly different for chlorophyll b in both fractions.

5. These results do not indicate that more than a single chlorophyll b complex occurs in vivo.     

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 μg/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 μg/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 μg/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.     

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy in order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure and conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules (the increased h₊₁/h₀ parameter of maleimide spin label, MSL; 0.377 ± 0.021 in diabetics vs 0.338 ± 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (τ_c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 ± 0.42 in diabetics, n = 21 vs 4.79 ± 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 ± 1.08, n = 28 vs 7.31 ± 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h₊₁/h₀ parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.     

Patterns of cartilage stiffness on normal and degenerate human femoral heads

Using the apparatus and technique described in an earlier paper, (Kempson et al. 1971), indentation tests were performed on areas of cartilage of 0·125 in dia., in situ on, and distributed evenly over, the cartilage surface of the human femoral head. Curves of indentation vs. time were plotted for a physiological stress of approximately 400 lbf/in². (28·2 kgf/cm², 2·36 MN/m²). The stiffness of each area of cartilage was calculated from the appropriate indentation value, in the form of a creep modulus at 2 sec, using the equations described in the previous paper (see Appendix). Layered maps and histograms showing the variation of both cartilage stiffness and indentation are presented.     

Fluorescence polarization and pigment orientation in photosynthetic bacteria

Fluorescence polarization of photosynthetic bacteria with various types of chromatophores suggests an orientation of bacteriochlorophylls, but not of carotenoids. Comparison of the in vitro fluorescence polarization spectra of bacteriochlorophyll at high and low concentration and at 77 °K with those in vivo at various temperatures and in the presence of carbowax indicates that dichroism of shape effects the in vivo spectra. Both orientation and shape effects are highest for bacteria containing lamellar-type chromatophores, and lowest in those containing vesicle-type ones.

The polarization values of the bacteria studied are similar for the various red bands, indicating a nearly parallel orientation of the adjacent bacteriochlorophylls.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Dominant role of interface over knee angle for cushioning impact loading and regulating initial leg stiffness

For in vivo impact loadings administered under controlled initial conditions, it was hypothesized that larger initial knee angles (IKA) and softer impacting interfaces would reduce impact loading and initial leg stiffness. A human pendulum was used to deliver controlled impacts to the right foot of 21 subjects for three IKA (0, 20 and 40°) and three interfaces (barefoot, soft and hard EVA foams). The external impact force and the shock experienced by the subjects' shank were measured simultaneously with a wall mounted force platform and a skin mounted accelerometer, respectively. Stiffness of the leg was derived using impact velocity and wall reaction force data. The results disproved the role of the knee joint in regulating initial leg stiffness and provided only partial support for the hypothesized improved cushioning. Larger knee flexion at contact reduced impact force but increased the shock travelling throughout the shank. Conversely, softer interfaces produced sizable reductions in both initial leg stiffness and severity of the impact experienced by the lower limb. Force rate of loading was found to be highly correlated (r=0.95) to limb stiffness that was defined by the heel fat pad and interface deformations. These results would suggest that interface interventions are more likely to protect the locomotor system against impact loading than knee angle strategies.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Platelet Activating Factor Stimulates and Primes the Liver, Kupffer Cells and Neutrophils to Release Superoxide Anion

Platelet activating factor (PAF) is considered a key mediator in eliciting the immunologic and metabolic consequences of endotoxic shock and sepsis. Release of oxygen-derived radicals is one of the important and relevant actions of PAF. This study examines the direct and priming effects of PAF on superoxide anion release by perfused liver, isolated Kupffer cells and blood neutrophils. One hour after PAF infusion at a dose of 2.2 μ/kg body weight a significant amount of superoxide release (0.71 ± 0.01 nmol/min/g liver) was measured in the perfused liver compared with the control livers (0.2 ± 0.01). In the in vitro presence of either phorbol ester or opsonized zymosan, superoxide release following PAF treatment in vivo was significantly increased to 1.36 ± 0.2 and 4.29 ± 0.36, respectively. The administration of PAF receptor antagonist (SDZ 63-441) almost completely inhibited the release of this radical. Kupffer cells (KC1, KC2, KC3) and blood neutrophils isolated from PAF-treated rats were also primed for increased production when these cells were challenged in vitro by the activator of protein kinase C, opsonin-coated zymosan as well as the chemotactic factors, complement 5a and F-met-leu-phe. PAF added in vitro to the perfused livers, isolated Kupffer cells or neutrophils from normal animals stimulated the release of superoxide with or without the above agonists. The direct stimulatory effect of PAF on superoxide release was inhibited by the PAF receptor antagonist in vitro. The role of PAF in the LPS-induced superoxide release by the perfused liver was also examined by the administration of PAF antagonist in endotoxic rats. The antagonist inhibited the LPS-mediated superoxide release at 1 hr, but not at 3 hr post-treatment. These results indicate that PAF stimulates and primes the hepatic elements to release superoxide. PAF may be an important factor during the early phase of endotoxemia, while other bioactive substances may take over at a later phase. Therefore, PAF is a key mediator that can directly enhance the release of toxic oxygen-derived radicals which may contribute to organ failure during endotoxemia or sepsis.     

Direct mechanics assessment of elastic symmetries and properties of trabecular bone architecture

A method is presented to find orthotropic elastic symmetries and constants directly from the elastic coefficients in the overall stiffness matrix of trabecular bone test specimens. Contrary to earlier developed techniques, this method does not require pure orthotropic behavior or additional fabric measurements. The method uses high-resolution computer reconstructions of trabecular bone specimens as input for large-scale FE-analyses to determine all the 21 elastic coefficients in the overall stiffness matrix of the specimen, using a direct mechanics approach. An optimization procedure is then used to find the coordinate transformation that yields the best orthotropic representation of this matrix. The method is illustrated here relative to two trabecular bone specimens. The techniques developed here can be used to obtain a complete characterization of the mechanical properties of trabecular architecture. With the development of in vivo reconstruction techniques, even in vivo measurements will be possible.     

In Vivo Esr Studies of Antioxidant Activity on Free Radical Reaction in Living Mice Under Oxidative Stress

In vivo antioxidant activity seems to be quite complicate due to multiple interaction with biomaterials and differs from results by in vitro experiments. In vivo estimation of antioxidant activity is performed by measuring TBA reactive substances in blood or hydrocarbon gases in breath, but these systems do not measure free radical reaction but the final products of oxidative reaction. In the present study, we applied in vivo ESR to evaluate antioxidant activity by monitoring the redox reaction of nitroxide radical and clearly found that the nitroxide is very susceptible to oxidative stress in vivo and quite useful to evaluate antioxidant activity non-invasively.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Measurement of Lipid Peroxidation

Lipid peroxidation results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. The approach to the quantitative assessment of lipid peroxidation depends on whether the samples involve complex biological material obtained in vivo, or whether the samples involve relatively simple mixtures obtained in vituo. Samples obtained in vivo contain a large number of products which themselves may undergo metabolism. The measurement of conjugated diene formation is generally applied as a dynamic quantitation e.g. during the oxidation of LDL, and is not generally applied to samples obtained in vivo. Lipid hydroperoxides readily decompose, but can be measured directly and indirectly by a variety of techniques. The measurement of MDA by the TBAR assay is non-specific, and is generally poor when applied to biological samples. More recent assays based on the measurement of MDA or HNE-lysine adducts are likely to be more applicable to biological samples, since adducts of these reactive aldehydes are relatively stable. The discovery of the isoprostanes as lipid peroxidation products which can be measured by gas chromatography mass spectrometry or immunoassay has opened a new avenue by which to quantify lipid peroxidation in vivo, and will be discussed in detail.     

Formol-Saline as A Cell Conserving Medium in the Micronucleus Test

It is widely recognized that tests using mammalian cell sytems are essential for assessing mutagenic hazard (Ishidate and Yoshikawa 1980). The micre nucleus test (Von Ledebur and Schmid 1973, Schmid 1973) is a convenient in vivo technique to overcome the shortcomings of in vitro bacterial methods. However, this assay requires high quality smears, for the production of which the solution used to avoid cell damage is critical.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Synthesis and biological properties of substituted 1,4-dihydro-5-methyl-4-oxo-3-quinolinecarboxylic acids

A series of substituted 1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-4-oxo-3-quinoline carboxylic acids was synthesized and tested for their in vitro and in vivo antibacterial activity. The introduction of a methyl group at the 5-position of quinoline nucleus enhanced characteristically the antibacterial activity against Gram-positive bacteria, including Streptococcus pneumonia, which is a major pathogen in the respiratory tract infection, while retaining Gram-negative activity. Among them, 1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid hydrochloride (grepafloxacin) exhibited potent in vitro antibacterial activity against Gram-positive bacteria such as Streptococcus pneumoniae and high in vivo efficacy on the experimental systemic infections caused by the Gram-positive and -negative bacteria tested. It also showed a high distribution to the lung and bronchoalveolar lavage fluid in comparison to reference drugs and is now undergoing clinical evaluation.     

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O₂ and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5×10⁷ cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O₂ and glucose metabolism in the scaffold (20 mm width–35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04×10⁻³ mol/s/m³ for O₂ at 0.71 mL/min and 1.91×10⁻² mol/s/m³ for glucose at 0.35 mL/min.