Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.     

The effects of short-chain fatty acids on the neuronal membrane functions ofHelix pomatia. I. electrical properties

The effects of short-chain fatty acids on the membrane excitability, current-voltage (I-V) characteristics, and cell volume of Helix pomatia neurons were studied. 2-Decenoic acid (DA), having 10 carbon atoms in the hydrocarbon chain, suppressed the excitability of bursting neurons RPa1 (Sakharov and Salanki, 1969) for 30-60 min, while valeric acid (VA), having 5 carbon atoms, had no significant effect on excitability. DA had three different effects on the excitability of beating neurons: in some neurons DA suppressed excitability as in bursting neurons; in a second type of neuron DA had a negligible effect on excitability; and in the neuron located near RPa1 DA had a pentylentetrazol (PTZ)-like effect, i.e., it converted the discharge of the neuron from beating to bursting. DA decreased the peak value of the current, inducing a negative-resistance region in the I-V curve of the bursting neuron without any change in the level of the voltage at which the current reaches its maximal value. DA inhibited the hyperpolarization induced by activation of the Na+ pump, tested after preliminary enrichment of neurons with Na+ ions by incubation in a potassium-free solution for 20 min. DA caused a swelling of the neuron by about 10% which was independent of the Na+ pump. In all the above-mentioned cases VA had no significant effect.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Number of hits necessary for complement-mediated hemolysis

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.     

Prostaglandin stimulation of adenosine 3',5'-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cyclic AMP level in cultured chondrocytes were examined. Prostaglandin E1 at 0.4 to 30 microM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D2 and E2 also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E1. Prostaglandin A1 had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E1, D2 or E2 was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E1, E2, or D2 on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin and PTH were more than additive. Addition of an inhibitor of cyclic nucleotide phosphodiesterase with prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E1, E2, and D2 increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Fermentative production of 5′-purine ribonucleotide

Micrococcus sodonensis KY 3765 and Arthrobacter citreus KY 3155 were found capable of accumulating IMP in media supplemented with hypoxanthine as a precursor. High concentrations of phosphate and magnesium salts were required for high yields of IMP. Manganese deficiency in the media was also essential. Excessive Mn²⁺ effects were also seen in the IMP fermentation carried out with an adenineless mutant, of Cornynebacterium glutamicum. In M. sodonensis, R5P-like substances, 5-phosphoribose pyrophosphokinase and IMP pyrophosphorylase, were leaked out, of the cells grown in suboptimal Mn²⁺ levels. This excretion was inhibited by high levels of Mn²⁺. Such a phenomenon was not noted in A. citreus. An adenineless mutant (KY 7208) of Brevibacterium ammoniagenes was found to accumulate an appreciable amount of IMP. The chemical changes in this fermentation showed that, hypoxanthine was first produced de novo, excreted, and then reconverted into IMP by a salvage pathway. When hypoxanthine was added to 7208 culture, IMP yield was increased appreciably. In fact exogenous ¹⁴C-hypoxanthine was incorporated into ¹⁴C-IMP. Subsequent experiments showed that indeed Br. ammoniagenes ATCC 6872, a parent culture of KY 7208, was able to produce IMP, GMP, and AMP, in good yield from hypoxanthine, guanine, and adenine, respectively.     

Prostaglandin E2 induced changes in renal blood flow, renal interstitial hydrostatic pressure and sodium excretion in the rat

Prostaglandin E2, when infused into the renal artery of the dog, is a vasodilator and increases both renal interstitial hydrostatic pressure and sodium excretion. Similar studies in the rat, however, have been inconclusive. The present study examined the effect of prostaglandin E2 infusion into the renal interstitium, by means of a chronically implanted matrix, on renal blood flow, renal interstitial hydrostatic pressure and sodium excretion in the rat. Prostaglandin E2 was continuously infused directly into the kidney interstitium to mimic endogenous prostaglandin E2 production by renal cells. The maximum change in each of these parameters occurred when 10(-5) M PGE2 was infused. Renal blood flow increased from 4.70 +/- 0.91 to 5.45 +/- 0.35 ml/min (p less than 0.05) while renal interstitial hydrostatic pressure decreased from 3.9 +/- 0.4 to 2.6 +/- 0.5 mmHg (p less than 0.05) and fractional excretion of sodium decreased from 1.02 +/- 0.20 to 0.61 +/- 0.12% (p less than 0.05). Thus, the present study demonstrates that renal interstitial infusion of prostaglandin E2 increases total renal blood flow but decreases both renal interstitial hydrostatic pressure and urinary sodium excretion in the rat.     

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.