Anti-edematous effects of epinastine, cetirizine and its enantiomers in λ-carrageenan-induced edema in rat hind paw.

Urticaria is induced by the histamine released from mast cells which develops wheals (edema) as a visual feature. In clinical practice, second-generation histamine H1 -receptor blockers are routinely used as the first-line symptomatic treatment for urticaria. Nevertheless, not much research has directly examined the second-generation histamine H1-receptor blockers' ability to reduce edema. In this study, we directly evaluated the anti-edematous activities of three second-generation histamine H1-receptor blockers available in the market (epinastine hydrochloride, cetirizine hydrochloride, and levocetirizine hydrochloride) using a λ-carrageenan-induced footpad edema model. One hour before the induction of edema with 1% λ -carrageenan injection, all second-generation histamine H1 -receptor blockers (5, 10, 50 and 100 mg/kg) were subcutaneously administered to rats. At 0.5 and 3 hours after λ -carrageenan administration, the edema volume was evaluated using a Plethysmometer. Epinastine hydrochloride significantly suppressed the edema growth in a dose-dependent manner. Cetirizine hydrochloride showed a slight anti-edematous effect, while levocetirizine significantly inhibited the development of edema in a dose-dependent manner. On the other hand, dextrocetirizine did not prevent edema from growing. In summary, second-generation histamine H1 -receptor blockers, at least those examined in this study, may be able to reduce the clinical symptoms of urticaria associated with edema. Levocetirizine hydrochloride is also anticipated to have stronger anti-edematous effects than cetirizine hydrochloride because levocetirizine is responsible for cetirizine's anti-edematous activity.

Direct comparison of anti-inflammatory effects of 14-, 15-, and 16-membered macrolide antibiotics in experimental inflammation model induced by carrageenan in rats.

Some macrolide antibiotics, which share a basic lactone ring structure, also exhibit anti-inflammatory actions in addition to their antibacterial activities. However, no study has directly compared anti-inflammatory effects on acute inflammation among macrolide antibiotics with the distinct size of the lactone ring. In this study, we evaluated and compared the anti-inflammatory activities of four 14-membered macrolides (erythromycin, clarithromycin, roxithromycin, oleandomycin), one 15-membered macrolide (azithromycin), and three 16-membered macrolides (midecamycin, josamycin, leucomycin) using a rat carrageenan-induced footpad edema model. All macrolide antibiotics were intraperitoneally administered to rats one hour before the induction of inflammatory edema with 1% λ -carrageenan. The anti-inflammatory effects on acute inflammation were evaluated by changing the edema volume. All 14-membered and 15-membered macrolide antibiotics significantly suppressed the development of edema. Conversely, none of the 16-membered macrolide antibiotics inhibited the growth of edema. In conclusion, compared to 16-membered macrolide antibiotics, 14-membered and 15-membered macrolide antibiotics have stronger anti-inflammatory effects. Further research should be done to determine why different lactone ring sizes should have distinct anti-inflammatory effects.

Anti-Inflammatory Actions of Expectorants in a Rat Carrageenan-Induced Footpad Edema Model.

S-Carboxymethyl-L-cysteine (SCMS) exhibits sputum-regulating and anti-inflammatory actions. Previous studies reported the anti-inflammatory effects of SCMS on chronic inflammatory diseases, but no study has examined these effects on acute inflammatory diseases. In this study, we investigated the anti-inflammatory effects of SCMS in a rat carrageenan-induced footpad edema model, which is routinely used as an acute inflammation model. Expectorants were administered to rats with footpad edema induced by subcutaneously administering 1%λ-carrageenan to the footpad of the left posterior limb, and the dose dependency of the anti-inflammatory effects was evaluated. As a result, even when the dose of SCMS was increased to 400 mg/kg, there were no inhibitory effects on edema. Furthermore, we examined the inhibitory effects of other expectorants (ambroxol hydrochloride, N-acetyl-L-cysteine, L-cysteine ethylester hydrochloride, and L-cysteine methylester hydrochloride), which were reported to exhibit anti-inflammatory effects on chronic inflammation, on edema. However, none of these expectorants inhibited edema.

Earthworm (Pheretima communissima and Pheretima hilgendorfi) hemoglobins: giant assemblies and subunits.

The molecular architecture of hemoglobins and their subunits of the earthworms Pheretima communissima and Pheretima hilgendorfi was investigated. In both species, their s0.20,w of 60.8 S and D020,w of 1.80 . 10(-7) cm2 . s-1 corresponded to a molecular weight of 3.07 . 10(6). From electron microscopic observations, the overall structure of the hemoglobins was shown to be two superimposed hexagonal discs, each composed of six-membered constituents. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of both hemoglobins revealed the presence of five species of subunits with molecular weights of 13,000-14,000 (subunit 1), 27,000-28,000 (subunit 2), 30,000-31,000 (subunit 3), 33,000-34,000 (subunit 4) and approx. 52,000 (subunit 5), respectively, and the molar ratio of these subunits 1:2, 3, 4:5 was 2:3:3. If we consider this set of the subunits 1 to 5 as one unit, the molecular weight of this unit should be 2.7-2.8 . 10(5). This one unit, therefore, should be considered to represent one-twelfth the whole molecule with molecular weight of 3.07 . 10(6).

Influence of quaternary structure on the state of the haem in carp and human methaemoglobins studied by resonance Raman scattering.

the resonance Raman spectra of carp and human methaemoglobin (metHb) derivatives in the T and R quaternary structures were measured in the frequency regions of 1200-1700 cm-1 and 100-600 cm-1. Conversion of carp and human fluoro metHb's to the T structure was accompanied by a frequency shift of the Raman line at 348 cm-1 to lower frequency by 5 cm-1, but all other lines were unaffected by the R-T transition. As the 348 cm-1 line was assigned to a porphyrin mode (v8) involving a bending motion of peripheral groups, the frequency change may be attributable to a change in van der Waals' interaction between haem and globin. On addition of inositol hexaphosphate to carp azide metHb, the intensity ratios of Raman lines (cm-1/cm-1), 1588/1567, 1641/1606, and 349/377, decreased. This indicates a shift of the high-spin/low-spin equilibrium towards high-spin side, in agreement with the rise in magnetic susceptibility. No distinct change was detected for the Raman spectra of aquo and cyano met Hb's on the R-T transition, even in the lower frequency region.

Functional properties of the glycosylated minor hemoglobins A1a-1,A1a-2 and A1b. EPR evidence for increased stability of the low affinity quaternary structure and decreased susceptibility to organic phosphate.

Electron paramagnetic resonance (EPR) spectra of the glycosylated minor hemoglobins A1a-1, A1a-2, A1b and A1c and the major hemoglobin A0 in the nitrosyl form have been obtained in the absence and presence of inositol hexaphosphate. In the absence of inositol hexaphosphate, nitrosyl hemoglobins A1a-1, A1a-2 and A1b exhibited a triplet hyperfine structure centered at g = 2.009 which has been shown to be diagnostic of the low affinity (T) quaternary structure. Addition of inositol hexaphosphate to nitrosyl hemoglobins A0, A1c, A1b and A1a-2 developed a triplet hyperfine structure of the EPR spectra but the magnitude of the hyperfine was decreased in the order of hemoglobins A0, A1c, A1b and A1a-2. However, inositol hexaphosphate had essentially no effect on the EPR spectrum of nitrosyl hemoglobin A1a-1. The present results account qualitatively for the oxygen binding properties of these glycosylated minor hemoglobins in the framework of a two-state allosteric model.

Identical myoglobin is present in both skeletal and smooth muscles of chicken.

Chicken gizzard has been considered to be an exceptional organ of smooth musculature in which a myoglobin is present. Since the characterization of the gizzard myoglobin has to date, been very incomplete, we studied the structures and functions in detail. The main component, which constituted roughly 90% of the protein, isolated by chromatofocusing, was homogeneous by electrophoretic and ultracentrifugal analyses. The molecular weight was consistently 1.8 X 10(4) by equilibrium sedimentation and iron analysis, and the isoelectric point was 7.8. Spectroscopic properties of the oxy-, carboxy- and deoxy-derivatives were typical of myoglobin. The oxygenation equilibria were also typical of myoglobin, showing neither homotropic nor heterotropic allosteric interactions, and the temperature-dependence (delta H0) was estimated as -16.6 kcal/mol. All these characteristics of the gizzard myoglobin were identical with those of the protein from the skeletal muscles. The amino acid composition and peptide mapping results also concluded that identical myoglobin was present in the gizzard, skeletal and probably cardiac muscles.

The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer.

The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0).

Preparation of native alpha and beta subunits from canine hemoglobin.

Preparation of native alpha and beta subunits from non-primate hemoglobins has never been successful using the procedure of Bucci, E. and Fronticelli, C. ((1965) J. Biol. Chem. 240, PC551-552). We describe a method for isolating the constituent subunits from canine hemoglobin. Human-canine hybrid hemoglobins (alpha 2Can beta 2A and alpha 2A beta 2Can) were prepared by acid hybridization techniques and DEAE-Sephadex chromatography. The hybrids were then reacted with an excess of p-chloromercuribenzoate under slightly acid conditions and the resultant alpha Can and beta Can subunits were isolated by either anion- or cation-exchange chromatography. Identifications of the subunits were made by gel electrophoresis and amino acid analysis. After removal of the bound mercurials with beta-mercaptoethanol, both subunits exhibited two reactive sulfhydryl groups per chain and were found to be fully in the native form, as judged by spectroscopy. By ultracentrifugal analysis, the alpha subunit was shown to be in a dimer-monomer equilibrium while the beta subunit was largely in a tetrameric form.

Occurence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.

[Complete resection for giant thymic carcinoma after simultaneous combination chemotherapy].

A 19-year-old man visited our hospital complaining of dyspnea. Chest X-ray and computed tomography (CT) showed a huge mass in the right anterior mediastinum. We diagnosed this as invasive thymoma by microscopic examination of specimens obtained by echo-guided needle biopsy. The patient underwent 6 courses chemotherapy [1st course : carboplatin (CBDCA) + doxorubicin hydrochloride (DXR) + vincristine sulfate (VCR) + cyclophosphamide (CPA), 2nd, 3rd-6th course : cisplatin (CDDP) + ADM + VCR + CPA]. At achievement of partial response (the reduction rate of the tumor size : 91.4%), the tumor was completely resected. The pathological examination of the resected specimens yielded a diagnosis of large cell carcinoma. Preoperative chemotherapy with ADOC regimen may be effective in advanced thymic carcinoma.

Enhancement of erythroid differentiation in clones of human leukemic cell line K562 by fetal calf serum.

Clone cells of K562 that are able to synthesize hemoglobin spontaneously on a relatively high level were obtained by cell-cloning technique. The clone cell proliferated 25 times by day 6 in culture, and the growth rate was not affected by changing the dose of fetal calf serum (FCS) from 5% to 30%. On the other hand, the erythroid differentiation could be linearly enhanced by increasing dosage of FCS, reaching a maximum after four days in culture. The wild-type K562 cells were also slightly stimulated to synthesize hemoglobin by adding FCS (30% final concentration). The enhancing effect of 30% FCS on the erythroid differentiation in the clone cells was greater than that of 12.5 microM hemin, while in the wild-type cells the relationship was reversed. There were no effects of erythropoietin (Epo) on the hemoglobin synthesis in either the clone cells or the wild-type cells. When various kinds of sera were added to the standard culture of the clone cells, only FCS had the enhancing effect. These results suggest that spontaneous erythroid differentiation is not induced by hemin or Epo in FCS but by FCS-specific substance(s).

Effects of cadmium on in vitro and in vivo erythropoiesis: erythroid progenitor cells (CFU-E), iron, and erythropoietin in cadmium-induced iron deficiency anemia.

Effects of cadmium (Cd) on in vitro and in vivo erythropoiesis in rats were studied by methylcellulose colony assay. Cd suppressed the in vitro growth of late erythroid progenitors (CFU-E) in a dose-dependent fashion and did not lose its inhibitory potency with increasing doses of erythropoietin (EPO). In addition, in marrow suspension cultures, Cd did not significantly influence 59Fe incorporation into both the cells and heme, and the Cd dose-responsive inhibition curve of the number of living cells was similar to that of CFU-E. These results suggest that the suppression of CFU-E colony formation by Cd is not due to the blocking of either EPO action to stimulate the growth of CFU-E or the iron incorporation into the cells ahd heme, but due to its direct cytotoxic effect. The colony suppression by Cd could be prevented by adding metallothionein to the cultures. On the other hand, oral administration of Cd to animals (100 mg/liter in drinking water) induced an iron deficiency anemia characterized by microcytic hypochromic red cells, decreased plasma iron, and increased total iron binding capacity. Marrow CFU-E density steadily increased as plasma iron decreased due to Cd administration and reached a plateau after 50 days. Plasma EPO titers were also found to be elevated in such a Cd-induced anemia. Parenteral iron administration during the Cd drinking period could completely prevent the development of iron deficiency anemia and the increase of both CFU-E and plasma EPO. There was a hyperbolic correlation between CFU-E and plasma iron or transferrin saturation. These results demonstrate that oral CD administration produces bone marrow hyperplasia at the CFU-E level due to iron deficiency.

Plasma erythropoietin assay by a fetal mouse liver cell culture method with special reference to effective elimination of erythroid colony inhibitor(s) in plasma.

Methods for the elimination of an inhibitor(s) of erythroid colony formation from plasma were examined in an attempt to measure genuine plasma erythropoietin (Epo) activities with an erythroid colony-forming assay using fetal mouse liver cells. Acid-boiling-chloroform (ABC) treatment was concluded to be the best method because the plasma thus treated stimulated colony formation most and contained the least protein. The dose-response curve for the plasma was parallel to that for the standard Epo preparation. The "erythroid colony-stimulating activity" in the plasma was completely additive to that in the standard Epo, and appeared to be a relatively heat-stable and acid protein with an isoelectric point lower than 5.0. These results suggest that the activity in the plasma is identical to that in the standard Epo. Stability of the plasma Epo activity was dependent on storage temperature and enhanced by adding 1% bovine serum albumin (BSA). Average Epo titers for normal adult, full-term cord, and murine plasmas, all ABC-treated and with 1% BSA added, were 192.4, 184.5, and 150.6 mU/ml, respectively. These values were much higher than those measured by the in vivo standard polycythemic mouse assay.

Further improvement in preparation and some properties of native alpha and beta chains from canine hemoglobin treated with p-chloromercuribenzoate.

The alpha and beta chains were prepared from canine hemoglobin by a modification of the method of Bucci and Fronticelli ((1965) J. Biol. Chem. 240, PC551-552) which involved treatment of hemoglobin with an excess of p-chloromercuribenzoate (pCMB), separation of the mercurated chains by chromatography on a DE-32 column with a salt gradient at pH 8.6, and regeneration of sulfhydryl groups of the chains with 2-mercaptoethanol. The SH titer was two per heme for both the regenerated alpha and beta chains. The titer decreased to four per tetramer of hemoglobin after equimolar recombination of both chains. Measurements of the absorption spectrum and oxygen binding showed that the chains were in a native state.

Flow-dependent influence of high-O2-affinity erythrocytes on peak VO2 in exercising muscle in situ.

To evaluate the influence of a high-O2 affinity of the erythrocyte and of flow rate on muscle's ability to extract O2 and develop force, we perfused dog gastrocnemius contracting isometrically at 4 Hz with normal-O2-affinity perfusate or high-O2-affinity perfusate at high and moderate flows (200 and 100 ml . min-1 . 100g-1, respectively). High-O2-affinity perfusate was prepared by incubating human citrate-phosphate-dextrose-stored erythrocytes with buffered saline containing cyanate (4 degrees C, 18 h) and normal-affinity perfusate by storing 2,3-diphosphoglycerate-rejuvenated erythrocytes in the same solution without cyanate. PO2 when blood is half oxygenated was 30.6 Torr for normal perfusate and 18.1 Torr for high-affinity perfusate. During 4-Hz stimulation, the tension developed by the muscle increased incrementally (positive staircase) to reach a peak value after 1.2-1.6 min for the normal perfusate and 0.6-0.7 min for the high-affinity perfusate (P < 0.05). The rate of decline during the early fatigue (measured from the onset of tension decline to 3 min) with high-affinity perfusate was significantly faster than it was with normal perfusate (P < 0.05). These findings suggest that both the staircase effect and the early fatigue are related to O2 availability, which is restricted when erythrocytes have a high O2 affinity. The peak O2 uptake values measured at 3 and 5 min were significantly lower (by 14-24%) with high-affinity perfusate than with normal perfusate at a given level of O2 delivery (arterial O2 content x flow) (P < 0.05). PO2 of venous effluent was proportionally related to peak O2 uptake. The present results indicate that neither blood flow nor O2 delivery is the sole determinant of the muscle's ability to extract O2.

Isolation and properties of myoglobins from rat (Rattus norvegicus) skeletal muscles.

One major (Mb I) and two minor (Mbs II and III) myoglobins, were isolated by a heat-denaturation-gel-filtration-chromato-focusing procedure from aqueous extract of rat skeletal muscles. The primary structure of the major component, as determined by an automatic Edman degradation method, revealed amino acid replacements at nine positions compared with murine myoglobin. As in murine myoglobin, a cysteine residue, highly reactive to p-chloromercuribenzoate (PMB), was present at position 66. The two minor components, with higher anodic mobilities and with SH group unreactive to PMB, were found to be derived from Mb I through mixed disulfide formation with cysteine (Mb II) or glutathione (Mb III) at the Cys66 residue. In view of the hydropathy profile, secondary structures of rat and mouse myoglobins showed few differences from each other. The three rat myoglobins displayed essentially identical oxygen equilibrium properties with neither homotropic nor heterotropic allosteric interactions, which agreed very well with those computed from the kinetic measurements.

Oxygen affinities (P50) of myoglobins from four vertebrate species (Canis familiaris, Rattus norvegicus, Mus musculus and Gallus domesticus) as determined by a kinetic and an equilibrium method.

Rate constants of the reaction with oxygen of myoglobins from four vertebrate species (Canis familiaris, Rattus norvegicus, Mus musculus and Gallus domesticus) and the isolated alpha A and beta A chains of human adult hemoglobin (HbA) were determined by the stopped-flow-spectrophotomeric method. Half-saturation oxygen pressure (P50) of the proteins calculated from the rate constants, assuming a simple bimolecular reaction model, agreed very well with those directly determined by oxygen equilibria. The proteins used were freshly prepared, and fully characterized by electrophoretic and ultracentrifugal analyses. Sulphydryl groups in the Hb chains were ascertained to be completely regenerated.

Mixed disulphide formation in myoglobin of mouse (Mus musculus).

The myoglobin, isolated from murine skeletal muscles by chromatofocusing, showed the three components, one major and two minor, with different electrophoretic mobilities. The major component with the isoelectric point (pI) of 7.55 had one reactive SH/mole, while the others with pI of 7.32 and 7.16 showed none, which could be rendered fully reactive by treating the proteins with beta-mercaptoethanol. The three components were the same in their molecular weight (18 kDa), amino-acid composition with one Cys residue and oxygenation properties. By a sensitive high-performance liquid chromatography method, the occurrence of cysteine or glutathione mixed disulphide was verified in the two minor components. We conclude from these results and incubation experiments with low-molecular-weight thiols that the two minors were derived from the major by a mixed disulphide formation with either cysteine or glutathione of the cysteinyl SH at the 66th sequence.

Oxygenation properties and intraerythrocytic constituents of human blood when stored in different media of ACD and CPD.

In the blood stored in acid-citrate-dextrose solution (ACD blood), the oxygen affinity and red cell 2, 3-diphosphoglycerate (2, 3-DPG) content showed parallel exponential decays with half-lives of 3 to 4 days. In the blood stored in citrate-phosphate-dextrose solution (CPD blood), the two parameters increased during the first 4 days before showing the same decay as that seen in the ACD blood. There was no significant change in the transmembrane pH gradient of the red cells, and thus the intracellular pH at the plasma pH of 7.40 was always in the range of 7.17 +/- 0.02 throughout the period of storage in ACD medium. In both ACD and CPD blood, the Hill exponent n was always normal (approximately 2.8) while the Bohr coefficient (delta log P50/delta pH) rose along with the lapse of time for preservation. The oxygen affinity of the CPD blood was less influenced by the red cell 2, 3-DPG than was that of the ACD blood. This phenomenon was thought to derive from higher concentration of salts within the CPD-stored red cells. The efficiency of blood oxygen transport in ACD and CPD blood was compared.