Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.     

Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.     

Thin-layer cytology findings of small cell carcinoma of the lower female genital tract. Review of three cases with molecular analysis

OBJECTIVE: To describe the thin-layer cytology findings of small cell carcinoma of the low female genital tract, with histologic correlation and human papillomavirus (HPV) genotyping. STUDY DESIGN: The authors reviewed the clinical findings, thin-layer cytology and histologic features of small cell carcinoma of the lower female genital tract (cervix or vagina) occurring in three postmenopausal Chinese women at Pamela Youde Nethersole Eastern Hospital, Hong Kong, over a four-year period, from January 1998 to December 2001. Molecular techniques for HPV screening and genotyping using the polymerase chain reaction and restriction fragment length polymorphism were employed on the cytologic specimens. RESULTS: The thin-layer preparations were of moderate to high cellularity. There were loose aggregates of or isolated small round cells with a high nuclear/cytoplasmic ratio, thin but irregular nuclear membrane, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm. Tumor cell cannibalism was commonly found. Small groups of tumor cells with nuclear molding were noted. There was also obvious tumor diathesis in the background. The necrotic debris was admixed with isolated small round cells, apoptotic bodies and nuclear dust. Associated koilocytosis or squamous intraepithelial lesions were not seen. Histologic examination of the tumor biopsies showed classic features of small cell carcinoma associated with squashing artifacts and vascularized stroma. Molecular analysis revealed the presence of HPV DNA (either type 18 or 16) in all the three liquid-based cytology samples. CONCLUSION: While the cytomorphologic features of small cell carcinoma of the cervix or vagina in thin-layer preparations are slightly different from those in conventional smears, due mainly to the absence of smearing effect, recognition of the subtle but characteristic appearance can enhance the accuracy of the cytologic diagnosis. The association between HPV and primary small cell carcinoma of the lower female genital tract was confirmed by this study.     

Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method

I report herein two 2.0 ns (1.0 fs time step) MD simulations of two zinc complexes bridged by a hydroxide in phosphotriesterase (PTE) employing the nonbonded method and the cationic dummy atom method that uses virtual atoms to impose orientational requirement for zinc ligands. The cationic dummy atom method was able to simulate the four-ligand coordination of the two zinc complexes in PTE. The distance (3.39 +/- 0.07A) between two nearby zinc ions in the time-average structure of PTE derived from the MD simulation using the cationic dummy atoms matched that in the X-ray structure (3.31 +/- 0.001A). Unequivocally, the time-average structure of PTE was able to fit into the experimentally determined difference electron density map of the corresponding X-ray structure. The results demonstrate the practicality of the cationic dummy atom method for MD simulations of zinc proteins bound with multiple zinc ions. In contrast, a 2.0 ns (1.0 fs time step) MD simulation using the nonbonded method revealed a striking difference in the active site between the X-ray structure and the time-average structure that was unable to fit into the density map of PTE. The results suggest that caution should be used in the MD simulations using the nonbonded method.     

Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Changes of adrenomedullin and receptor activity modifying protein 2 (RAMP2) in myocardium and aorta in rats with isoproterenol-induced myocardial ischemia

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2). To explore the pathophysiological roles of adrenomedullin and its receptor component RAMP2 in ischemic cardiovascular diseases, we studied the changes of adrenomedullin and RAMP2 mRNA in myocardium and aorta in rats with isoproterenol (ISO)-induced myocardial impairment. In ISO-treated rats, heart became enlarged markedly, the ratio of heart to body weight was increased by 54% (P<0.01), and myocardial malondialdehyde content and plasma lactate dehydrogenase activity was elevated by 43% (P<0.01) and 138% (P<0.01), respectively. Immunoreactive adrenomedullin (ADM) in plasma, myocardium and aorta was augmented by 116.7% (P<0.01), 50.8% (P<0.01) and 12.5% (P>0.05), respectively. ADM mRNA in myocardium and aorta was increased by 96.8% (P<0.01) and 38.5% (P<0.01), respectively. RAMP2 mRNA in myocardium and aorta was increased by 19.6% (P<0.05) and 15.8% (P<0.01), respectively. These results suggest that the increase of ADM level and the up-regulation of ADM and RAMP2 gene in myocardium and aorta may be significant in the pathogenesis of ischemic myocardiopathy.     

Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 x 10(-10) M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 x 16 cm) were perfused with Krebs buffer equilibrated with 95% O(2) and 5% CO(2) at 37 degrees C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 x 10(-10) M) significantly amplified the concentration-dependent (10(-7)-10(-5) M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10(-8)-10(-6) M) skin vasoconstrictor effect of the thromboxane A(2) mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10(-7) M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca(2+) in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 x 10(-10) M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 x 10(-6) M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca(2+) in smooth muscle cells.