The characteristics of dorsal-root ganglia and sensory innervation of the hip in rats.

Using a rat model the characteristics of the sensory neurones of the dorsal-root ganglia (DRG) innervating the hip were investigated by retrograde neurotransport and immunohistochemistry. Fluoro-Gold solution (FG) was injected into the left hip of ten rats. Seven days later the DRG from both sides between T12 and L6 were harvested. The number of FG-labelled calcitonin gene-related peptide-immunoreactive or isolectin B4-binding neurones were counted. The FG-labelled neurones were distributed throughout the left DRGs between T13 and L5, primarily at L2, L3, and L4. Few FG-labelled isolectin B4-binding neurones were present in the DRGs of either side between T13 and L5, but calcitonin gene-related peptide-immunoreactive neurones made up 30% of all FG-labelled neurones. Our findings may explain the referral of pain from the hip to the thigh or lower leg corresponding to the L2, L3 and L4 levels. Since most neurones are calcitonin gene-related peptide-immunoreactive peptide-containing neurones, they may have a more significant role in the perception of pain in the hip as peptidergic DRG neurones.

Synthesis and bioactivities of novel 5,6-bis(4-methoxyphenyl)-2H-pyridazin-3-one derivatives: inhibitors of interleukin-1 beta (IL-1beta) production.

New 5,6-bis(4-methoxyphenyl)-2H-pyridazin-3-one derivatives were prepared, and their abilities to inhibit IL-1beta production were evaluated. Some compounds showed potent inhibitory activity against IL-1beta production in HL-60 cells stimulated with lipopolysaccharide (LPS). The synthesis and structure-activity relationships of these compounds are described.

Synthesis and bioactivities of novel pyridazine derivatives: inhibitors of interleukin-1 beta (IL-1beta) production.

New pyridazine derivatives were prepared, and their abilities to inhibit IL-1beta production were evaluated. Some compounds showed potent inhibitory activity against IL-1beta production in HL-60 cells stimulated with lipopolysaccharide (LPS). The synthesis and structure-activity relationships of these compounds are described.

Adverse reactions to radiocontrast media in an Indian population.

This study was conducted to determine the epidemiological characteristics and clinical presentation of adverse reactions to intravenous radiocontrast media (CM) in patients of Indian origin. 379 of 1798 patients who received either sodium iothalamate or sodium meglumine diatrizoate developed adverse reactions (i.e. 21.08% of patients). The incidence of mild, moderate and severe adverse reactions were 19.47%, 1.33% and 0.28%, respectively. One patient who developed a severe reaction expired. There were no differences in the incidence of adverse reactions according to gender (males 21.2%; females 20.8%; p = 0.907) or age (p = 0.876). The incidence of adverse reactions was significantly higher in patients with a history of previous reactions (45.5%) than in those with no history (20.9%; p = 0.046). The incidence of reactions was also significantly higher in patients with a history of predisposing factors such as bronchial asthma (69.2%) and diabetes mellitus (60.0%) than in those without such a history (20.6%; p = 0). The incidence of adverse reactions in patients who received premedication prior to CM administration because of a history of predisposing factors (21.4%) was not significantly different from that in patients who were not premedicated (21.2%; p = 0.974), a result probably due to inadequate premedication used in the study. The skin was the most commonly affected site of reaction.

Problem in diagnostic imaging: pancreaticoduodenal arcade in splanchnic arterial stenosis.

This paper features a problem in diagnostic imaging in which a pre-transplant abdominal angiogram of a potential liver recipient shows filling of the hepatic artery via the superior mesenteric artery and the pancreaticoduodenal arcade. The routing of this unusual supply to the liver is explained by careful study of abdominal aortic angiograms and a sagittal MRI made through the aorta.

Increased anticoagulant activity of recombinant thrombomodulin modified with glycosaminoglycan.

Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activated protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.

Importance of sialic acid in recombinant thrombomodulin in terms of pharmacokinetics and separation of desialyzed glycoprotein.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.

Transjugular liver biopsy: a comparison of aspiration and trucut techniques.

The results of 67 transjugular liver biopsies are described. Two failures were encountered due to inability to pass the needle into acutely angulated hepatic veins. Thirty-four patients underwent a liver aspiration biopsy using a Colapinto needle, while the remainder were biopsied using a trucut needle. The success rate with the Colapinto needle was 68% and with the trucut model, 97%. Capsular perforation occurred in three cases, but without significant morbidity or mortality. It is concluded that the trucut needle biopsy is more reliable than aspiration biopsy, when the transjugular approach is mandated, in obtaining optimal liver tissue for histopathological diagnosis.

Morphology of the corpus callosum in human fetuses.

Shape and size of the human fetal corpus callosum of a relatively racially homogeneous southern Indian sample population were studied in midsagittal sections of formalin fixed brains. Length of corpus callosum and width of its genu, body, and splenium were measured and the data statistically analyzed. Presence of an isthmus between the body and splenium did not correlate with the measured variables. There was no significant gender difference. The variables correlated significantly among each other but only callosal length and genu width correlated with gestation age. Significant absolute increase occurred in callosal length and genu width, whereas body and splenium widths remained the same. Simple regression equations to estimate the callosal length and genu width for a given age are derived.

Digestion of endothelin-1 on cultured vascular smooth muscle cells.

The degradation of 125I-endothelin-1 (125I-ET-1) was examined on cultured porcine aortic endothelial cell (EC) and rat vascular smooth muscle cell (SMC) by HPLC analysis. The degradation of ET-1 was observed on SMC and was slightly observed on EC. Membrane fractions of SMC had a strong potency for the degradation of ET-1 and retained activity even in plasma. This activity was inhibited with some of the noble inhibitors which were known as enkephalinase inhibitors. These results suggest that endothelin degradation enzyme on SMC is related to enkephalinase and that this enzyme plays a significant role in the degradation of ET-1 in vivo.

Purification and characterization of endothelin-1 degradation activity from porcine kidney.

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.

[Abnormal secretory response to verapamil of pancreatic B cells of neonatal rats maintained in high glucose].

The effect of verapamil on insulin secretion in the presence of IBMX was studied in B cells in pancreatic monolayer cultures of the neonatal rat using a perifusion system. For the initial three days, B cells were exposed to a medium of either 5.5 or 11.1 mM glucose and 10 microM iodoacetic acid (IAA) after which they were exposed to a medium of either 5.5 or 11.1mM glucose until day 7. Insulin secretion of B cells in medium of 5.5mM glucose (day 7) was not affected by IBMX, and the observations showed that verapamil significantly suppressed the secretion of insulin. However, the second phase of secretion was significantly increased by verapamil and IBMX. In contrast, insulin secretion of B cells exposed to a medium of 11.1mM glucose (day 7) showed similar trends, but the second phase secretion was increased further by verapamil in the presence of IBMX. The total amount of secretion was 2.3 times (125.5ng/ml) the secretion of cells exposed to low concentration of glucose (day 7). These results show that there is a possibility that in the presence of IBMX, verapamil, which is known to be a Ca2+ channel antagonist, shows the action of a Ca2+ channel agonist and promotes the secretion of insulin. Moreover, there were indications that a high concentration of glucose in culture affects functional maturation and functional differentiation of pancreatic B cells of neonatal rats and impairs the intra-cellular signal transduction system.

Pyrogenic action of endothelin in conscious rabbit.

Injection of 0.3 nmol/kg endothelin(ET)-1 into the ear vein of conscious rabbits induced a significant increase in body temperature. ETB receptor specific agonist, namely 4-Ala-ET-1, also caused an elevation of the body temperature in a dose-dependent manner by injection into the ear vein of rabbit. These results suggest that ET play important roles in regulation of body temperature through selective stimulation of ETB receptor.

Induction of dyspnea by aerosol of endothelin-1/histamine or/methacholine in the conscious guinea pig.

The inhalation of aerosol of endothelin-1 (ET-1) induced dyspneal behavior in conscious guinea pigs pretreated with histamine or methacholine inhalation, although it was not observed by the inhalation of ET-1 alone. The dyspneal response to the inhalation of ET-1/histamine was potently inhibited by indomethacin, but it was not affected by nifedipine or diphenhydramine.

Characterization of immunoreactive endothelin in human urine.

We developed three antibodies, specific and sensitive to endothelin-1 (ET-1), and established two sandwich and three competitive enzyme immunoassays (EIAs). By using these EIAs, large immunoreactive ET (IR-ET) of molecular weight 10 k Da was identified as a main component of IR-ETs in human urine. This large IR-ET, which reacted with two antibodies specific for N-terminal region of ET-1 but not with the antibody against C-terminal peptide of ET-1, was partially purified by six-step procedure and examined by Western blotting after SDS polyacrylamide gel electrophoresis. The large IR-ET was detected as a single band at molecular weight of 10 k Da both in reduced and non-reduced conditions. From these results, the large IR-ET was thought to consist of a single polypeptide chain and possess the steric restricted N-terminal region of ET-1.

Syntheses and biological activities of endothelin-1 analogs.

Endothelin-1 analogs replaced by various amino acids at position 21, namely [X21]-ET-1, were synthesized, and their agonistic vasoconstrictor activity on rat thoracic aortic strips and receptor binding activity on rat brain membrane fraction were examined to elucidate their structure-activity relationship. The vasoconstrictor activities of [Tyr21]- and [Phe21]-ET-1 were one order of magnitude smaller than that of ET-1, and those of [His21]-, [Gly21]-, [Ser21]-, [Ala21]- and [Lys21]-ET-1 were more than two orders of magnitude smaller than that of ET-1. On the other hand, the replacements by Ile, Glu, Gln and Pro resulted in distinguished losses of the vasoconstrictor activities. In addition, preincubation with these analogs did not blunt ET-1-induced vasoconstriction and showed no antagonistic activity. The binding inhibitory activities of these analogs against 125I-ET-1 were approximately conformable to the vasoconstrictor activities with only a slight exception. These findings demonstrate that the phenyl group at position 21 is important for both the vasoconstrictor activity and the receptor binding activity.

Inhibition of endothelin (ET)-1-and ET-2-induced vasoconstriction by anti-ET-1 monoclonal antibody.

We produced a monoclonal antibody to endothelin (ET)-1, tested cross-reactivities with the related peptides by enzyme immunoassay, and investigated the effects of the antibody on ET-1- or ET-2-induced vasoconstriction of rat isolated thoracic aorta. The antibody recognized ET-1, ET-2 and ET-3, and the immunoreactive site proved to be the N-terminal region but not the C-terminal region of ET-1. Moreover, at an approximate molar-equivalent concentration, the antibody absorbed ET-1 and ET-2, and significantly inhibited ET-1- and ET-2-induced vasoconstriction notwithstanding the presence of the endothelin receptor.

In vitro and in vivo antibacterial activities of K-4619, a new semisynthetic aminoglycoside.

The antibacterial activities of K-4619 (3-de-O-methylsporaricin A sulfate) were compared with those of sporaricin A, amikacin, dibekacin, and gentamicin. K-4619 exhibited a high order of activity against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa. Its activity against Providencia species and Serratia marcescens was the highest of all drugs tested. K-4619 was highly effective against bacteria that produce various aminoglycoside-inactivating enzymes, except for 3-acetyltransferase I. The bactericidal activity of K-4619 was somewhat greater than that of amikacin. The activity of K-4619 against gram-negative bacteria increased at alkaline pH and was hardly affected by inoculum size, addition of horse serum, and composition of the medium. The in vivo protective effect of K-4619 against infections with Klebsiella pneumoniae, S. marcescens, and P. aeruginosa in mice was greater than that of sporaricin A. K-4619 was also active in mice infected with gentamicin- or amikacin-resistant strains bearing some of the aminoglycoside-inactivating enzymes.

Antimicrobial and beta-lactamase inhibitory activities of carpetimycins A and B, new carbapenem antibiotics.

Carpetimycins A and B showed widely broad spectra and potent activity against gram-positive and gram-negative bacteria, including various species of anaerobic bacteria. The antimicrobial activity of carpetimycin A was 8 to 64 times greater than that of carpetimycin B and 4 to 128 times greater than that of cefoxitin. The inhibitory concentration of carpetimycin A required to inhibit more than 90% of clinical isolates was 0.39 micrograms/ml for Escherichia coli and klebsiella and 1.56 microgram/ml for Proteus and Staphylococcus aureus. At a concentration of 3.13 micrograms/ml, carpetimycin A inhibited almost all clinical isolates of Enterobacter and Citrobacter, which showed resistance to many clinically used beta-lactam antibiotics. Carpetimycins A and B furthermore were shown to have potent inhibitory activities against several kinds of beta-lactamases produced by beta-lactam-resistant strains; they inhibited not only penicillinase-type beta-lactamases but also cephalosporinase-type beta-lactamases, which were insensitive to clavulanic acid. In combination with beta-lactam antibiotics such as ampicillin, carbenicillin, and cefazolin, carpetimycins A and B showed synergistic activities against beta-lactam-resistant bacteria.

[Effect of prednisolone 17-valerate 21-acetate on immunological responses in mice (author's transl)].

The in vivo effects of prednisolone 17-valerate 21-acetate (PVA), an anti-inflammatory glucocorticoid on several immunological responses in mice were investigated in comparison with hydrocortisone 17-butyrate (HB) and betamethasone 17-valerate (BV), when given subcutaneously. PVA reduced the spleen weight, the number of splenic nucleated cells, the formation of hemolytic plaque forming cells (PFC), delayed type footpad reaction and the responsiveness of splenic lymphocytes to concanavalin A. These suppressive effects were almost the same as those seen with HB and weaker than those of BV. However, the responsiveness of splenic cells to lipopolysaccharide and circulating IgM antibody response to sheep red blood cells were suppressed by a smaller dose of PVA than that of HB. PVA had no effect on the responsiveness to phytohemagglutinin-P, whereas HB and BV enhanced the phytohemagglutinin-P responsiveness. The suppressive effect of PVA on the host defense to experimental infection with Escherichia coli was weaker than those of HB and BV. From these results, PVA appears to be similar to other glucocorticoids in that it exerts complicated effects on several immunological responses in mice.