Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia.

Fat body cells of silkmoth pupae (Hyalophora cecropia ) contain granules, showing a less dense outer zone and a denser, often crystalline, inner portion appear after cocoon spinning and increase until the larval-pupal ecdysis; more granules are formed in females than in males. Urate granules, appearing fibrous in internal structure, first form about the same time, but their accumulation is more gradual, and continues in the pupa. Both types have been isolated by centrifugation. Protein granules dissolve in buffers to yield proteins 1 and 2, with distinct electrophoretic and antigenic properties. These proteins have been isolated individually from pupal fat body extracts by using their different thermal stabilities in phosphate buffer containing MgCl2 and (NH4)2SO4, respectively, and purification was completed by gel chromatography. Protein 1 has a molecular weight of 480,000 and a subunit of 85,000 daltons, while protein 2 gives values of 530,000 and 89,000, respectively. Their amino acid compositions are similar but distinct. Proteins 1 and 2 accumulate in the hemolymph, beginning 3 days before spinning, reach maximal levels at spinning, and then decline in the hemolymph while granules are formed in the fat body, although the total hemolymph protein concentration does not decline at this time. It is concluded that the fat body of the late, feeding larva synthesizes two related "storage proteins" and secretes them in partially crystalline granules as protein reserves for metamorphosis.

[Preoperative workup to assess indication for laparoscopic treatment in gastroesophageal reflux disease].

INTRODUCTION AND OBJECTIVES: antireflux surgery performed by an experienced surgeon is a maintenance option for patients with well-documented gastroesophageal reflux disease (GERD). Well-documented GERD is difficult to find, as GERD is a multifactorial disease in which the gastroesophageal junction, with its special anatomical and functional components, is important. In order to examine patient preoperative workups, and their indication for surgical treatment in GERD, we retrospectively studied patients who underwent a laparoscopic antireflux procedure. METHODS: preoperative workups in patients from our health care area who underwent a laparoscopic antireflux procedure from December 1997 to February 2007 were retrospectively analyzed. Data related to epidemiological findings, symptoms, morphologic and functional evaluation, medical therapy, and indication for surgical treatment were recorded and statistically analyzed by means of a bivariate test. Differences were significant when the p value was equal to or less than 0.05. RESULTS: 100 patients (50 % female, 51.31 +/- 13.53 years of age) underwent a laparoscopic antireflux surgery after 56.47 +/- 61.33 months with symptoms. Ninety-five percent of patients had an anatomical abnormality. The pH monitoring test diagnosed three quarters of cases. The most frequent indication for GERD treatment was persistent or recurrent esophagitis despite adequate medical treatment (52 cases). CONCLUSIONS: based on our preoperative workup, as described, 100 percent of subjects were well documented and diagnosed with GERD (both non-erosive reflux disease and erosive reflux disease), and their indication for laparoscopic treatment was retrospectively assessed in 94% of cases.

Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme.

This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell-leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IXalpha, the product of HO-catalyzed degradation of protoheme IX. In these rats, oxidative stress such as superfusion with H(2)O(2) and ischemia-reperfusion of the tissues neither induced rolling nor exhibited adherent responses of leukocytes in venules. In contrast, the oxidative stresses evoked marked rolling and adhesion of leukocytes in the control rats without HO-1 induction. The HO-1 induction also downregulated leukocyte adhesion elicited by other pro-oxidant stimuli such as N(omega)-nitro-L-arginine methyl ester. The decreases in the oxidant-elicited leukocyte adhesive responses under HO-1-inducing conditions were restored by perfusion with zinc protoporphyrin-IX, an HO inhibitor, but not with copper protoporphyrin-IX, which did not inhibit the enzyme. Furthermore, the effects of zinc protoporphyrin-IX were repressed by superfusion with bilirubin or biliverdin at the micromolar level, but not by the same concentration of carbon monoxide, another product of HO. These results indicate that induction of the HO-1 activity serves as a potential stratagem to prevent oxidant-induced microvascular leukocyte adhesion through the action of bilirubin, a product of HO reaction.

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis.

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Cellular kinetics and histological changes in experimental cancer of the uterine cervix.

Carcinoma of the mouse uterine cervix was induced by the insertion of 20-methylcholanthrene (MC) thread into the uterine cavity. Through biweekly observations, it was seen that the histopathological carcinogenesis in the cervix was characterized by three distinct changes: normal epithelium, anaplastic epithelium, and carcinoma. The incidence rate of normal epithelium declined sharply in the first 4 weeks, followed by slight decline up to the 20th week of observation. Nevertheless, about 20% of MC-treated mice maintained histologically normal epithelium even at the 20th week. In contrast, the incidence rate of anaplastic epithelium increased sharply in the first 4 weeks and thereafter tended to decline. Carcinoma was first observed at the 4th week, and its incidence rate increased thereafter almost linearly, reaching about 50% of the MC-treated mice at the 20th week. Carcinoma was divided into two groups, the early invasive and the frank invasive types. The former was further subgrouped into epithelial bud, nodular growth, epithelial cord, and mixed type by the mode of stromal invasion. Studies using [3H]thymidine autoradiography on the cell proliferation kinetics in the process of carcinoma development showed that a labeling index became higher as the malignant changes of the epithelium advanced. Moreover, S-phase prolongation was observed with the malignant changes, and the cell cycle time did not differ markedly among normal epithelium (22.8 hr), anaplastic epithelium (23.0 hr), and frank invasive carcinoma (26.1 hr). However, the growth fraction was greatly varied; it was 28% for normal epithelium, 61% for anaplastic epithelium, 100% for early invasive carcinoma, and 100% for frank invasive carcinoma. These results indicate that the growth fraction increases as the malignant changes in the MC-treated mouse uterine cervix.

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides.

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.

Selectin on activated platelets enhances neutrophil endothelial adherence in myocardial reperfusion injury.

OBJECTIVES: The glycoprotein P-selectin is an adhesion molecule that is rapidly expressed on the surface of platelets and endothelium during the inflammatory process. P-selectin on endothelium has been reported to play an important role in reperfusion injury. However, little is known regarding P-selectin on platelets in contributing to the pathophysiology of myocardial reperfusion injury. In this study, we hypothesized that P-selectin on platelets may enhance neutrophil endothelial adherence and this may play a role in neutrophil-mediated reperfusion injury. METHODS: Endothelial cells, cardiomyocytes, platelets and neutrophils were isolated from adult rats. Endothelial cells and cardiomyocytes were cultivated in a co-culture system. After exposure to hypoxia and reoxygenation, neutrophil adherence and migration were examined. RESULTS: After exposure to 6 h of hypoxia, endothelial cells co-incubated with platelets showed significantly greater neutrophil adherence (63.1 +/- 4.0%) and migration (78.2 +/- 6.7%) than endothelial cells alone (adhesion: 44.2 +/- 2.8%, migration: 57.9 +/- 4.9%). These increases were significantly inhibited (adhesion: 42.1 +/- 3.5%, migration: 65.5 +/- 3.8%) by an anti-P-selectin monoclonal antibody. Moreover, the superoxide-anion production was significantly elevated when activated platelets were added to neutrophils. This enhanced production was also inhibited by anti-P-selectin antibody. CONCLUSION: The presence of activated platelets enhanced neutrophil adhesion and migration process after hypoxia reoxygenation. This process may occur following platelet-neutrophil interactions via P-selectin and subsequent neutrophil activation.

Growth hormone, prolactin and chorionic somatomammotropin in normal and molar pregnancy.

Twenty patients with molar pregnancy, ten normal pregnant women and ten healthy non-pregnant women were given 30 g of arginine intravenously. The serum concentration of growth hormone, prolactin and chorionic somatomammotropin (CS) was determined by radioimmunoassay. In addition, serum 17beta-estradiol, estriol and progesterone were also measured. Arginine infusion induced a sharp rise of GH in patients with molar pregnancy and in nonpregnant subjects, but the response in normal pregnancy was blunted. The response of PRL was high in patients with molar pregnancy, blunted in normal pregnancy and very small in nonpregnant subjects. CS did not respond at all to arginine infusion both in normal pregnancy and molar pregnancy. The high response to argine of PRL, normal response of GH and low baseline secretion and no response of CS may be characteristic of molar pregnancy.

Role of P-selectin in endotoxin-induced uveitis.

PURPOSE: P-selectin is one of the early-reactive adhesion molecules that play a part in the rolling phase of leukocytes in cellular infiltration. The study objective was to determine whether P-selectin is involved in the development of endotoxin-induced uveitis (EIU). METHODS: Endotoxin-induced uveitis was initiated in male Lewis rats by injecting 200 micrograms lipopolysaccharide (LPS) into the foot pad. Expression of P-selectin in the iris-ciliary body was studied by immunohistochemistry using wholemounts and paraffin embedded sections of iris-ciliary body prepared at various time intervals. A monoclonal antibody (mAb) against P-selectin and a ligand for P-selectin, sialyl Lewis X oligosaccharide (SLeX-OS), was intravenously injected to evaluate the effects of selectin inhibition. The effect of treatment was evaluated by the number of infiltrated cells and protein concentration in the aqueous humor at 24 hours after LPS treatment. RESULTS: P-selectin immunoreactivities were observed in the vessels of the iris in whole iris-ciliary body mounts and on the surface of the microvascular endothelium in paraffin-embedded sections. Activity was most prominent at 15 minutes and at 5 to 7 hours after LPS treatment and was moderate from 1 to 4 hours after treatment. The selective inhibition of P-selectin significantly blocked the cellular infiltration into aqueous humor, but this infiltration was even more effectively inhibited by SLeX-OS. Protein concentration in the aqueous humor was not inhibited by selectin as much as was cellular infiltration. CONCLUSIONS: In the early phase of EIU, P-selectin may be expressed on the vascular endothelium in the iris in a biphasic pattern that modulates the rolling phase of leukocytes. The expression of this molecule may be essential for succeeding processes of cellular infiltration and may determine the subsequent states of ocular inflammation.

Contribution of E-selectin to cellular infiltration during endotoxin-induced uveitis.

PURPOSE: The selectin family is a group of early-reactive adhesion molecules that plays a role in the rolling phase of leukocytes in cellular infiltration. It has been reported that P-selectin is expressed on vascular endothelium in the iris-ciliary body 15 minutes after lipopolysaccharide (LPS) treatment in endotoxin-induced uveitis (EIU) and may contribute to the initial phase of ocular inflammation. The objective of the present study was to identify the expression pattern of E-selectin, another member of the selectin family, and to investigate the role of E-selectin during the course of EIU. METHODS: Endotoxin-induced uveitis was induced in male Lewis rats by a footpad injection of 200 microg LPS. The time-dependent expressions of E-selectin in EIU in the iris- ciliary body and the retina were studied by immunohistochemistry using wholemounts and paraffin-embedded sections and by monitoring the level of E-selectin mRNA expression. A monoclonal antibody to E-selectin and a control antibody were each injected intravenously to evaluate the effects of E-selectin inhibition on ocular inflammation at the time of maximum uveitis. In the anterior uvea, the effect was evaluated by the number of infiltrated cells and by the protein concentration in the aqueous humor 24 hours after LPS treatment; in the retina, the myeloperoxidase (MPO) activity was measured 48 hours after LPS treatment. The effect of the combined injection of anti-P-selectin and anti-E-selectin antibodies was also studied. It was then determined whether the delayed inhibition of E-selectin (6, 12, or 24 hours after LPS injection) could contribute to the early resolution of the uveitis. RESULTS: E-selectin immunoreactivity was observed on the vessel walls of the iris and retina 7 hours after LPS treatment in wholemounts and paraffin-embedded sections and remained positive for 24 hours after LPS treatment. The expression of E-selectin messenger RNA gene peaked at 6 hours and again at 18 hours after LPS treatment in the iris- ciliary body and retina. The expression returned to the basal level 24 hours after LPS treatment in the iris- ciliary body and 48 hours after LPS treatment in the retina. The selective inhibition of E-selectin significantly blocked the cellular infiltration into the aqueous humor (P < 0.005) but had a milder effect on the protein concentration in the aqueous humor (P=0.0536). The inhibition of E-selectin and P-selectin almost abrogated cellular infiltration (P < 0.001). Myeloperoxidase activity in the retina 48 hours after LPS treatment was again significantly decreased by the inhibition of E-selectin alone and by the inhibition of E-selectin and P-selectin (P < 0.0001). A single injection of anti-E-selectin antibody 6, 12, or 24 hours after LPS injection effectively blocked cellular infiltration in the aqueous humor 24 and 48 hours after LPS treatment. CONCLUSIONS: In EIU, E-selectin may be expressed on the vascular endothelium and remain after the period of expression of P-selectin and until approximately the time of maximum uveitis. The present results suggest that, in contrast to the role of P-selectin as an initiator of cellular infiltration, E-selectin may contribute to continuing cellular infiltration into the inflammatory site during inflammation, and its inhibition may contribute to the early resolution of the uveitis.

In vivo evaluation of platelet--endothelial interactions after transient retinal ischemia.

PURPOSE: Accumulating evidence suggests that platelets play an important role in ischemia-reperfusion injury. To fulfill that role, platelets flowing in the bloodstream would have to interact with retinal endothelial cells and to accumulate in the postischemic retina. This study was designed to investigate quantitatively platelet-endothelial interactions in postischemic retina after transient retinal ischemia. METHODS: Transient retinal ischemia was induced in Long-Evans rats for 60 minutes by temporal ligation of the optic nerve. Isolated platelet samples labeled with carboxyfluorescein diacetate succinimidyl ester were administered intravenously to recipient rats after various reperfusion periods. Platelet-endothelial interactions in postischemic retina were evaluated in vivo with a scanning laser ophthalmoscope. Anti-P-selectin monoclonal antibody (mAb) was administered 5 minutes before the injection of labeled platelets. P-selectin gene expression in the postischemic retina was studied by semiquantitative polymerase chain reaction. RESULTS: Under basal conditions, infused platelets showed minimal interactions with retinal endothelial cells. In contrast, postischemic retinas showed active platelet-endothelial interactions. Many platelets were observed rolling along and adhering to the major retinal veins. The number of rolling and adhering platelets reached a peak (555 +/- 65/mm per min and 25.8 +/- 3.2/mm(2)) 12 hours after reperfusion. However, the interactions between platelets and postischemic retinal endothelial cells were substantially inhibited by neutralizing P-selectin expressed on endothelial cells. In addition, P-selectin gene expression in postischemic retina corresponded with the time course of platelet-endothelial interactions during the reperfusion period. CONCLUSIONS: This study demonstrated that platelets actively interacted with retinal endothelial cells in the postischemic retina through P-selectin expressed on the retinal endothelial cells.

Retinal ischemia-reperfusion injury attenuated by blocking of adhesion molecules of vascular endothelium.

PURPOSE: To evaluate quantitatively the effects of blocking of adhesion molecules (P-selectin or intercellular adhesion molecule-1 [ICAM-1]) on leukocyte dynamics in the retinal microcirculation in vivo during ischemia-reperfusion injury and the therapeutic efficacy of the blocking of adhesion molecules on retinal ischemia-reperfusion injury. METHODS: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic nerve. P-selectin or ICAM-1 monoclonal antibody (mAb) was administered at 5 minutes before reperfusion. At 4, 12, and 24 hours after onset of reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 or 14 days of reperfusion, retinal damage was evaluated histologically. RESULTS: P-selectin mAb significantly inhibited leukocyte rolling along the major retinal veins after reperfusion. Subsequently, the number of accumulated leukocytes decreased in the P-selectin-inhibited rats. ICAM-1 mAb also inhibited leukocyte accumulation during the reperfusion period in a more substantial manner than P-selectin mAb. Histologic examination demonstrated the protective effect of the blocking of P-selectin or ICAM-1. In accordance with a reduction in leukocyte accumulation, the protective effect of mAb on retinal ischemia-reperfusion injury was more substantial in ICAM-1-inhibited rats. CONCLUSIONS: The present study demonstrates the inhibitory effect of P-selectin and ICAM-1 mAb on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.

In vivo evaluation of platelet-endothelial interactions in retinal microcirculation of rats.

PURPOSE: This study was designed to develop a new method to evaluate the dynamics of platelets in the retinal microcirculation in vivo and to investigate quantitatively the platelet-endothelial interactions in rat retina with the use of this system. METHODS: Isolated platelet samples were labeled with carboxyfluorescein diacetate succinimidyl ester. After intravenous administration, platelet behavior in the retinal microcirculation was evaluated with a scanning laser ophthalmoscope. The images were recorded on S-VHS videotape and analyzed with a computer-assisted image analysis system. The platelet- endothelial interactions in the retinal microcirculation were also investigated with the use of lipopolysaccharide-stimulated endothelium or platelets activated with thrombin. RESULTS: Fluorescent platelets were recognized as distinct dots in the retinal microcirculation and could be traced frame by frame. The velocity of platelets in the retinal arteries, capillaries, and veins was 26.1+/-6.4, 1.6+/-0.4, and 19.9+/-8.2 mm/sec, respectively. In control rats, even the activated platelets showed minimal interaction with retinal endothelial cells. In contrast, stimulated retinal endothelium showed active platelet- endothelial interactions; many platelets were observed rolling and adhering along the major retinal veins. The interactions between platelets and stimulated endothelial cells were substantially inhibited with the injection of P-selectin monoclonal antibody. CONCLUSIONS: The present study demonstrated a new method to visualize platelet behavior in the retinal microcirculation in vivo. This method will allow quantitative evaluation of platelet dynamics and platelet- endothelial interactions in retinal pathologic conditions.

Plasminogen activator in rat ovary during the ovulatory process: independence of prostaglandin mediation.

To elucidate the possible roles of increased plasminogen activator (PA) in follicular rupture and to investigate whether prostaglandins participate in ovarian PA synthesis in vivo, enzyme activities were sequentially measured by a method using the chromogenic substrate S-2251 in immature rat ovaries primed with pregnant mare serum gonadotrophin (PMSG) followed by human chorionic gonadotrophin (hCG) either alone or with a concurrent injection of indomethacin. Before hCG injection PA activity was 0.006 +/- 0.006 (S.D.) mumol/1.6 mg ovarian tissue (wet wt) per 30 min: PA activity of a saline-treated group remained at low levels (less than 0.018 +/- 0.003 mumol/l X 6 mg tissue per 30 min). In contrast, PA activity of animals given hCG alone increased after the treatment, reaching a peak value of 0.112 +/- 0.071 mumol/l X 6 mg tissue per 30 min 12h later, before decreasing to 0.023 +/- 0.014 mumol/l X 6 mg tissue per 30 min at 32 h. Contrary to expectations, a dose of indomethacin which completely blocked ovulation had no effect on either the magnitude or the time-course of PA synthesis after hCG administration (P greater than 0.05). These results indicate that prostaglandins are not involved in the preovulatory synthesis of PA induced by hCG in rat ovaries and that PA is not a primary proteolytic enzyme for follicular rupture. It is suggested that PA has possible roles in cumulus detachment and/or proliferation of granulosa cells during the ovulatory process.

Sequence homology of rat and human HCNP precursor proteins, bovine phosphatidylethanolamine-binding protein and rat 23-kDa protein associated with the opioid-binding protein.

The hippocampal cholinergic neurostimulating peptide (HCNP) enhances acetylcholine synthesis in rat medial septal tissues. We have cloned the cDNAs of the precursor proteins of rat and human HCNP and deduced their respective amino acid sequences. The HCNP sequences aligned at the N-terminal regions of their precursors. The deduced amino acid sequences showed homology with those of the bovine brain phosphatidylethanolamine-binding protein and the rat protein associated with the opioid-binding protein. These observations suggest that the HCNP precursor proteins may have multiple functions.

Mechanism of expression of the rat HCNP precursor protein gene.

The hippocampal cholinergic neurostimulating peptide (HCNP), isolated from hippocampal tissue of 10- to 12-day-old rats, enhances the in vitro synthesis of acetylcholine in medial septal tissue explants. The HCNP precursor is a 21 kDa protein that binds hydrophobic ligands and Mg-ATP, and is associated with the opioid-binding protein. We employed an HCNP-precursor cDNA as probe to clone the genomic DNA, used for mapping of the exon-intron structure of the gene. We also determined the nucleotide structure of the promoter region of the rat HCNP precursor protein gene. By using S1 mapping and CAT as a reporter, we found multiple promoters that were aligned in the 5' untranslated region. In addition, the presence of several putative enhancer binding sequences were tested by electrophoresis mobility shift assays. Northern blot analysis revealed that the gene is expressed in a variety of rat tissues and various subregions of the brain. These results suggest that HCNP-precursor gene expression is regulated by a general transactivation factor such as SP1, and that the specific presence of the bioactive HCNP in certain tissues results from post-translational events such as proteolytic processing of the precursor protein, which takes place predominantly in the hippocampus of young rats.

P-selectin participates in cardiopulmonary bypass-induced inflammatory response in association with nitric oxide and peroxynitrite production.

OBJECTIVES: P-selectin participates in the development of inflammatory disorders. Cardiopulmonary bypass is thought to induce inflammatory response and increase nitric oxide production. To evaluate the role of P-selectin in bypass-induced inflammatory response and its association with nitric oxide production, we examined the effect of P-selectin monoclonal antibody in a rat model of cardiopulmonary bypass. METHODS: Twenty adult Sprague-Dawley rats undergoing cardiopulmonary bypass for 60 minutes were divided into 2 groups. A 3-mg/kg dose of anti-rat specific P-selectin monoclonal antibody (ARP2-4; Sumitomo Pharmaceuticals, Osaka, Japan) was administered into the priming solution before bypass in group P (n = 10) and a 3-mg/kg dose of PNB1.6 (nonblocking monoclonal antibody) was added in group C for control (n = 10). RESULTS: At the termination of bypass and 3 hours after the termination of bypass, plasma levels of interleukins 6 and 8, nitrate/nitrite, the percentage ratio of nitrotyrosine to tyrosine (an indicator of peroxynitrite formation), and the respiratory index were significantly higher than before bypass in both groups, and they were significantly lower in group P than in group C. Plasma P-selectin level in group C and exhaled nitric oxide concentration in both groups at termination of bypass were significantly lower than those before bypass, and they were significantly higher 3 hours after termination of bypass than before bypass in both groups. Plasma P-selectin level and exhaled nitric oxide concentration in group P were significantly higher than those in group C at the end of bypass, but they were significantly lower 3 hours after the termination of bypass. CONCLUSIONS: These results demonstrate that P-selectin may participate in the augmentation of bypass-induced inflammatory response in association with nitric oxide and peroxynitrite production.

Characterization of glycoproteins isolated from porcine zonae pellucidae.

Two glycoproteins, tentatively designated as PZ-alpha and PZ-beta, have been isolated and purified to homogeneity from porcine zonae pellucidae by a simple purification procedure producing a high yield. The procedure included the dissolution of the zona material in 0.1 M sodium borate buffer pH 10.0, Sephadex G-100 column chromatography and preparative SDS-polyacrylamide gel electrophoresis. The purified glycoproteins gave a single band on polyacrylamide gel and had molecular weights of 60 000 (PZ-alpha) and 96 000 (PZ-beta). Glutamic acid was detected as the NH2-terminal residue in both glycoproteins, using the dansyl chloride method. Though their amino acid compositions were similar, their carbohydrate contents were slightly different (PZ-alpha: 24.9%; PZ-beta; 19.6%), but these components contained the same types of monosaccharides: fucose, mannose, galactose, NAcGlc and sialic acid. The antigenic properties of the two glycoproteins were indistinguishable by immunodiffusion tests. The PZ-beta could be converted in part to smaller molecular weight components, though not to PZ-alpha, by treatment with beta-mercaptoethanol. Thus clear differences between PZ-alpha and PZ-beta could not be detected by chemical or immunological analyses except for the difference in the behaviour on SDS-polyacrylamide gel electrophoresis.

Role of selectin-dependent adhesion in cardiac allograft rejection.

BACKGROUND: Selectins play important roles in the inflammatory responses by eliciting leukocyte rolling. The roles of E- and P-selectins in the acute rejection of cardiac allografts remain unclear. This study was designed to evaluate whether E- and P-selectins participate in the pathophysiology of heart rejection. METHODS: Heterotopic heart transplantation was performed in both mice and rats in full histoincompatibility combinations. Immunohistochemistry, flow-cytometry, and reverse transcriptase- polymerase chain reaction were performed to evaluate E-, P-selectin and sialyl Lewis X (SLeX) expression in rejecting cardiac allografts. The effects of short-term administration of monoclonal antibodies (mAbs) to E- and P-selectins on cardiac allograft survival were also evaluated. RESULTS: Significant prolongation of graft survival was observed in mice treated with either anti-E- or P-selectin mAbs, or both. The enhanced endothelial and mRNA expression of E- and P-selectins was observed in the rejecting cardiac allografts. Some graft- infiltrating mononuclear cells were double-stained with both anti-SLeX and anti-alphabetaT cell receptor mAbs. Flow-cytometric analysis of graft-infiltrating cells also showed enhanced SLeX expression. CONCLUSION: These results suggest that both P- and E-selectins are critically involved in the early development of acute heart rejection.

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.