Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B

Grossmann, Gertie, Yasuhiro Suzuki, Bengt Robertson, TsutomuKobayashi, Per Berggren, Wen-Zhi Li, Guo-Wei Song, and Bo Sun.Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B. J. Appl.Physiol. 82(6): 2003-2010, 1997.Near-termnewborn rabbits were exposed via the airways to a monoclonal antibodyto surfactant protein B and ventilated for 0-120 min. Controlanimals received nonspecific rabbit or mouse immunoglobulin G, saline,or no material via the airways. Administration of the antibody at 40mg/kg elicited an immediate, significant fall in lung-thorax complianceassociated with progressive intra-alveolar edema and/oralveolar collapse and necrosis and desquamation of airway epithelium,and hyaline membranes. The vascular-to-alveolar leak of human albuminand human immunoglobulin G, injected intravenously at birth anddetermined in lung lavage fluid 60-120 min after instillation ofthe antibody, was 1.8% for the left lung, with no difference betweenthe markers. The average leak in control animals ventilated for 120 minwas <0.3% (P < 0.05). Cytospin preparations of lung lavage fluid from animals exposed to the antibodyshowed significantly increased recruitment of neutrophilic granulocytes. The pathology and pathophysiology of neonatal lung injuryinduced by the monoclonal antibody to surfactant protein B probablyreflect a combination of direct inactivation of surfactant and aninflammatory response triggered by the immune reaction.

     

Reversed micelles recognize an active protein

Summary The extraction behavior of native and heated-denatured -chymotrypsin has been investigated with two different reversed micellar systems. A large difference in the degree of extraction was observed for the native relative to the denatured -chymotrypsin. In particular, mixed reversed micelles formulated with DOLPA (dioleyl phosphoric acid) and AOT show a high selectivity for the active -chymotrypsin.     

Does Coinfection of Bartonella henselae and FIV Induce Clinical Disorders in Cats?

It was found that Bartonella henselae (B. henselae) may induce clinical disorders in cats in natural conditions from a comparison of the serological status for B. henselae with the serostatus for feline immunodeficiency virus (FIV) and several clinical characteristics in 170 domestic cats. Seropositivity for B. henselae was not significantly different between FIV antibody-positive and -negative cats (18.4% vs 16.0%). The incidence of clinical characteristics were compared among four cat groups distinguished by the reactivity of sera against B. henselae and FIV. The incidence of lymph node swelling was lower in only FIV antibody-positive cats (3.0%), but higher in B. henselae antibody-positive cats (13.6%) and significantly higher in both B. henselae and FIV antibody-positive cats (42.9%) compared with the incidence of lymph node swelling in cats which were negative for both antibodies (5.5%). The same relation was also observed for the incidence of gingivitis among the 4 cat groups, suggesting that coinfection of B. henselae and FIV may be associated with gingivitis and lymphadenopathy in cats.     

Mutations of the Btk gene in 12 unrelated families with X-linked agammaglobulinemia in Japan

Alterations of the Bruton's tyrosine kinase(Btk) gene are responsible for X-linked agammaglobulinemia (XLA). Although mutations in various regions were reported mainly in the Caucasian population, correlation between the locations of mutation and the clinical phenotypes remains unclear. We report 12 abnormalities of theBtk gene found in 12 unrelated families out of 14 XLA families in Japan and their clinical features. We utilized Southern blotting and single-strand conformation polymorphism (SSCP) analysis. Gene rearrangement in the kinase domain was identified in two patients by Southern blotting. Seven point mutations, two small deletions, and one small insertion were detected by SSCP and sequencing. The SSCP analysis also provided information about the carriers in these families. We found some clinical heterogeneity in the affected family members with the same gene mutation. Moreover, there is considerable inconsistency between the locations of gene aberrations and the immunological phenotypes. Some patients with a nonsense mutation, which may result in the lack of kinase domain, have detectable B cells and immunoglobulins. These identified alterations will provide valuable clues to theBtk protein function and the pathogenesis of XLA.     

Characterization of the human p57KIP2 gene: Alternative splicing,insertion/deletion polymorphisms in VNTR sequences in the coding region,and mutational analysis

We have isolated human cDNA and genomic clones of a gene termed p57^KIP2, which is related to the p21^WAF1 and p27^KIP1 genes that encode inducible inhibitors of cyclin-dependent kinase activity. The p57 gene contains three GC-rich introns of 166 bp, 566 bp, and 83 bp, and two of the four exons correspond to coding regions. Alternative splicing generates the heterogeneity in the translational initiations. As this gene has been localized to chromosomal band 11p15.5, a region thought to be the location of a tumor suppressor gene(s) for carcinomas of the breast, bladder, and liver, we have examined a large number of tumors for genetic alterations of p57. Although no somatic mutation has been detected, we have found several normal variations in this gene, including four types of 12-bp in-frame deletions in the proline/alanine repeating domain, in which nearly 40 motifs, viz., 5′-CCGGCC-3′, are tandemly repeated. Received: 9 August 1995     

Structural Regions of the Cardiac Ca Channel α1C Subunit Involved in Ca-dependent Inactivation

We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel. Transfection of HEK293 cells with the wild-type α_1C or its 3′ deletion mutant (α_1C−3′del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of α_1C involved in this process, we analyzed chimeric α₁ subunits in which one of the major intracellular domains of α_1C was replaced by the corresponding region from the skeletal muscle α_1S subunit (which lacks Ca-dependent inactivation). Replacing the NH₂ terminus or the III–IV loop of α_1C with its counterpart from α_1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of α_1C with the corresponding region from α_1S dramatically slowed the inactivation of Ba currents while preserving Ca-dependent inactivation. A similar but less pronounced result was obtained with a II–III loop chimera. These results suggest that the I–II and II–III loops of α_1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of α_1C with the corresponding region from α_1S completely eliminated Ca-dependent inactivation without affecting inactivation of Ba currents. Significantly, Ca-dependent inactivation was restored to this chimera by deleting a nonconserved, 211–amino acid segment from the end of the COOH terminus. These results suggest that the distal COOH terminus of α_1S can block Ca-dependent inactivation, possibly by interacting with other proteins or other regions of the Ca channel. Our findings suggest that structural determinants of Ca-dependent inactivation are distributed among several major cytoplasmic domains of α_1C.     

Sesquiterpenoids in two different kinds of agarwood

Sesquiterpenoids of an agarwood originating from Aquilaria agallocha and of the other kind of agarwood (Aquilaria sp.; probably Aquilaria malaccensis) were investigated by a combination of GLC and GC/MS. The differences in sesquiterpene composition between the two kinds of agarwood are discussed.     

Changes in Conformation and Stability upon SCF/sKit Complex Formation

Stem cell factor (SCF) is thought to be a member of the four-helical bundle cytokine superfamily, and exists in solution as a noncovalent homodimer. It is the ligand for Kit, a tyrosine kinase type III receptor. The interaction of SCF and Kit affects early hematopoietic progenitors, as well as gametocytes, melanocytes, and mast cells. Upon binding of SCF the Kit undergoes dimerization and transphosphorylation. Circular dichroism (CD), intrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy were used for conformational analyses of free SCF, soluble Kit (sKit), and the complex. The sKit consisted of the extracellular domain of Kit, contained five Ig-like domains, and was prepared from the conditioned media of transfected Chinese hamster ovary cells. With these techniques, a reproducible conformational change was seen upon ligand/receptor binding. The far-UV CD and FTIR spectroscopy indicated a slight increase in the -helical content. The near-UV CD and fluorescence spectra showed changes in the environments of the aromatic amino acids. The thermal denaturation of SCF was not affected by complex formation, while the melting temperature of sKit increased only a few degrees when binding SCF. This indicates that binding is temperature dependent, consistent with titration calorimetry results published previously which demonstrated that there is a large enthalpy of binding. The conformational changes which accompany SCF/sKit binding could play a role in the receptor dimerization and signal transduction which follow.     

Purification and immunohistochemistry of Griffonia simplicifolia agglutinin-II-binding mucus glycoprotein in rat stomach

Gastric mucus is thought to protect the gastric wall from mechanicaltrauma, desiccation, pathogenic microorganisms, acid and proteases.We purified Griffonia simp1icifolia agglutinin-II (GSA-II)-bindingmucus glycoprotein (GMG) from rat gastric mucosa by solubilizationin a guanidine- containing buffer, gel permeation chromatography,Ricinus communis agglutinin-I (RCA-I)-affinity chromatographyand GSA-II-affinity chromatography. Rat GMG showed high molecularweight on a Sephacryl S-1000 column, and a single band in 0.5%agarose-2% polyacrylamide composite gels and blots. A proteinof {small tilde}60 kDa was contained in the GMG preparation.GMG was deglycosylated with trifluoromethanesulphonic acid treatment.An antibody was raised against deglycosylated GMG (deGMG). Theantibody recognized deGMG, GMG, periodic acid-treated deGMGand O-glycanase-digested deGMG, but did not react to trypsin-digesteddeGMG. These results suggest that the antibody recognizes proteinase-sensitiveregion or peptide backbone of GMG. In immunohistochemistry,the mucous gel layer of the stomach luminal surface was stainedwith antibody. The antibody recognized not only gastric mucousneck cells and pyloric gland cells, but also gastric surfacemucous cells, mucous cells in the duodenal gland, and gobletcells in the small intestine and colon. These results indicatethat GMG is a component of rat gastric mucus, and that the antibodyrecognizes mucous-secreting cells in rat stomach and intestine. antibody immunohistochemistry lectin-affinity gel chromatography mucus glycoprotein rat stomach     

A New Nitric Oxide (No) Releaser: Spontaneous No Release from Fk409

The remarkable vasorelaxant and anti-platelet effects of FK409 have been reported to be due to nitric oxide (NO) release. The purpose of the present study is to investigate the spontaneous NO-releasing pathway of FK409 in aqueous solutions. 'H-NMR spectra of FK409 suggested that the compound underwent a time-dependent elimination of the hydrogen atom at a-position of the nitro moiety (at the 5-position) in weakly alkaline solutions. In addition, the degradation of FK409 monitored by HPLC showed a pH-dependency accelerating with an increase of pH. These results revealed that the first step in the degradation of FK409 might be the hydroxyl ion-dependent subtraction of the hydrogen atom at the 5-position. On the other hand, NO release from FK409 also exhibited a pH-dependency, and the velocity of NO liberation was markedly enhanced above pH 6. Furthermore, a linear relationship between the rate of FK409 degradation and that of NO formation was observed, indicating that the rate-limiting step for NO formation is the same as that for degradation. Thus, the rate-limiting process of NO formation from FK409 is due to the deprotonation reaction of the hydrogen atom at the 5-position by hydroxyl ions. The deprotonation process appears to be an essential step for both FK409 degradation and NO release. On the basis of the results, a possible kinetic scheme for NO release from FK409 is proposed.