Histochemical changes in neonatal liver caused by vaginal instillation of magnetic nanoparticles in pregnant mice

Drug delivery through the vagina is a novel and effective approach for treating embryonic diseases. Magnetic nanoparticles (MNPs) currently are used as drug delivery systems. The safety of MNPs for use with embryonic tissues remains unclear. We used pregnant mice to investigate the possible toxicity of MNPs toward neonatal liver at three embryonic ages using histochemical and immunohistochemical techniques. MNPs were instilled through the vaginas of pregnant mice at days 12 (E12), 15 (E15) and 17 (E17) after fertilization. We found MNPs in the neonatal liver parenchyma after delivery of the pups on day 20. We observed that MNPs caused mild apoptosis of hepatocytes, cytoplasmic vacuolation and lymphocytic infiltration in the neonatal liver after treatment at E15 compared to instillation at E12 and E17. We observed also that MNPs increased the production of caspase proteins and tumor necrosis factor receptor 2 proteins, which are indicators of apoptosis, in the neonatal liver after instillation of MNPs at E15 compared to instillation at E12 and E17. MNPs also increased the number of collagen fibers and the amounts of connective tissue growth factors in the neonatal liver parenchyma after instillation at E15 compared to instillation at E12 and E17. The general carbohydrates in the neonatal liver were decreased in a time-dependent manner after instillation at E17, E15 and E12 owing to the presence of MNPs in the parenchyma. Overall, we determined that MNPs were mildly toxic to neonatal liver.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Reaction properties of hydrophobic enzymes and their immobilization on phospholipid composite membranes

Reaction rates of hydrophobic enzymes, aminopeptidase, and alkaline phosphatase, in microsomes prepared from the porcine brush border membrane and in vesicles pre pared from microsomes and phospholipids were measured at various temperatures. Interactions between the hydrophobic enzymes and the phospholipid layers are discussed as well as the effects of fluidity change of phospholipid layers on enzyme activity. Further, reaction properties and stabilities of the immobilized vesicles containing microsomal enzymes were studied.     

Depressant action of Ca-antagonists on slow action potentials in guinea pig ventricular muscles

The depressant action of four Ca antagonists, including a novel drug, tiapamil, on Ca channels was investigated using a conventional microelectrode technique. "All or none" slow action potentials were recorded in K+-depolarized guinea-pig papillary muscles. Verapamil and diltiazem decreased the amplitude and maximum rate of rise (Vmax) of the slow action potentials at concentrations up to 2 microM. The depressant effect of a novel Ca-antagonist, tiapamil, on the slow action potentials was as marked as that of verapamil and diltiazem. However, prenylamine was less potent than the other 3 drugs. In addition, the action of all drugs on the slow action potentials was enhanced as the frequency of stimulation was increased between 0.0083 and 1 Hz. It was concluded that tiapamil, as verapamil and diltiazem, produced a frequency-dependent blockade of the slow Ca channel.     

Reactivation processes of three inward current systems involved in the rising phase of the action potentials in embryonic chick hearts

In embryonic chick hearts during development, there are three inward current systems which are involved in the rising phases of the action potentials (APs): fast INa, slow ICa, and tetrodotoxin-insensitive slow INa. To assess reactivation processes for these three types of inward current channels (fast Na+, slow Ca2+, and slow Na+ channels), diastolic recovery of Vmax was examined in embryonic chick hearts using a paired-pulse protocol. In all cases, the diastolic recoveries were approximated by single exponential functions. The time constants of recovery (tau(V)) and T90% (the diastolic interval which allows 90% recovery of Vmax of the premature AP) were, respectively, 53.1 +/- 5.2 and 61.5 +/- 8.6 ms for Na+-dependent fast AP (n = 10), 376.9 +/- 49.3 and 659.2 +/- 113.1 ms for the Ca2+-dependent slow AP (n = 10), and 40.7 +/- 5.3 and 45.6 +/- 12.0 ms for the Na+-dependent slow AP (n = 10). In the presence of lidocaine, the recovery kinetics also appeared to be single exponentials for diastolic intervals up to 500 ms (fast APs) or 250 ms (slow APs). The reactivation processes for the Na+-dependent fast and slow channels were significantly slowed by 100 microM lidocaine. In addition, in the presence of 100 microM lidocaine, Vmax was depressed in a frequency-dependent manner; the higher the stimulation frequency, the greater the depression. Hence, the fast Na+ channels and the slow Na+ channels had the following similarities: rapid reactivation, reactivation slowed by lidocaine, and frequency-dependent depression in the presence of lidocaine.     

Arachidonic acid production by the red alga Porphyridium cruentum

The single-celled alga, Porphyridium cruentum, was assessed by means of chromatographic separation and mass spectral analysis of its fatty acids to be a potentially competetive source of arachidonic (5,8,11,14-eicosatetraenoic) acid. Models for both cell growth and production of the prostaglandin precursor at various temperatures and light intensities are presented. Increasing the light intensity within the range 1700-8000 lux increases the cell growth rate without affecting the arachidonic acid yield per cell; increasing the cultivation temperature from 18 degrees C to ca. 32 degrees C lowers the yield of arachidonic acid per cell but increases the rate of its production per unit volume and time. The increase of the weight ratio of arachidonic:palmitic acids at low temperatures is interpreted as a means of controlling the microviscosities of cellular membranes. In addition, the arachidonic acid content of cells decreases with the culture's age, despite increases in unit cell dry weight. The maximum rate of 0.46 mg arachidonic acid L(-1) h(-1) was calculated by means of the model to occur at ca. 32 degrees C and 8000 lux in liquid cultures of 12 x 10(9) cells/L. Estimates of the cost of producing arachidonic acid by means of this alga range from $0.15/g to $1.00/g of arachidonic acid. Cells grown at 18 degrees C in the presence of 0.3% linoleic acid swelled and produced gorlic (13-cyclopent-2-enyltridec-6-enoic) acid and another compound not normally observed. An estimated threefold increase of arachidonic acid content also occurred, but no significant lipogenesis was induced at 23 degrees C in the presence of 1% kerosene or 0.3% palmitic, stearic, oleic, or linoleic acids.     

Application of porous teflon tubing method to automatic fed-batch culture of microorganisms II. Automatic constant-value control of fed substrate (ethanol) concentration in semibatch culture of yeast

An automatic feedback control system incorporating a porous Teflon tubing sensor was developed and a strain of yeast was cultivated semibatchwise in mineral salt medium by feeding pure ethanol as the sole carbon source. In the control system, The ethanol concentration was continuously measured by the porous Teflon tubing sensor combined with a flame ionization detector, and its output signals were furnished to an automatic feed controller which controlled an ethanol feed pump so that deviations from the set level of ethanol concentration might be corrected. The controller was constructed on the basis of proportional-differential negative feedback control of which the proportional sensitivity and differentiation constants were estimated from the dynamic mass balance of ethanol. Precise measurement of temperature and compensation of the detector output signals for temperature fluctuations of culture broth were necessary to achieve good control. Cultivation experiments were carried out with three levels of concentrations: 10², 10³, and 10⁴ ppm. The relative deviations of the concentrations were less than ±0.5% for the 10³- and 10⁴- ppm levels but a little offset arose for the 10²-ppm levels. The growth of cells was at first exponential and then almost linear when the dissolved oxygen concentration dropped considerably.     

Incorporation and activation of a membrane-bound enzyme in bilayers of liposomes

Summary The effects of lipid composition and fluidity of lipid bilayers on incorporation and activation of membrane-bound d-fructose dehydrogenase are described in this study. The incorporation of the enzyme into bilayers of small unilamellar vesicles (SUV) made of several phospholipids resulted in enzyme activation with magnitudes higher than that observed in the presence of Triton X-100, indicating that this higher activation is due to lipid-protein interaction. The activity was highest in the presence of SUV formed by the addition of 10% dl--dipalmitoylphosphatidylethanolamine to l--dimyristoylphosphatidylcholine, which resulted in eightfold higher activation compared with that of the enzyme in its free state. This activation did not appear to be due to the degree of incorporation of the enzyme, indicating that incorporation is distinct from the activation event. Thus, it is probably the lipid environment that leads to higher activation of the enzyme. A break in the Arrhenius plot of the activity of the membrane-bound enzyme at temperatures close to the phase transition of the phospholipid implies that changes in the physical state of the lipid bilayer influence the enzyme activity. Furthermore, immobilization of d-fructose dehydrogenase, previously adsorbed to SUV, on urethane prepolymer also resulted in about eightfold higher activation than that of the free enzyme. Offprint requests to: S. Katoh     

Effects of prostaglandin F2 alpha and prostacyclin on pulmonary microcirculation in the cat

In pulmonary microcirculation, using a new X-ray television system, we measured the effects of prostaglandin F2 alpha (PGF2 alpha) and prostacyclin on the internal diameter (ID), flow velocity, volume flow, and transit times of a contrast medium in small arteries (Ta) and veins (Tv) in anesthetized cats. The ID of the arteries and veins ranged from 100 to 500 micron. PGF2 alpha, 0.3, 1, and 3 micrograms/kg, predominantly decreased ID on the arterial side in a dose-dependent manner but increased flow velocity 27-62%. Consequently, volume flow was kept relatively constant. With PGF2 alpha, Ta and Tv were decreased 18-41% and 4-15%, respectively. Prostacyclin, 2 and 4 micrograms/kg, uniformly dilated the ID of small arteries 9-16% but did not change small veins. With prostacyclin, flow velocity was unchanged or decreased, whereas volume flow was increased significantly, 27-32%. No significant changes of Ta and Tv were observed in response to prostacyclin. When both prostaglandins, PGF2 alpha and prostacyclin, were administered, they canceled each other with respect to the ID of small pulmonary arteries. Prostacyclin also prevented the PGF2 alpha-induced vasoconstriction of the pulmonary venous microcirculation.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.