Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion

A very sensitive time-resolved fluoroimmunoassay for the prothoracicotropic hormone (PTTH) of the silkworm Bombyx mori has been established. The lower limit of detection in this assay was 0.1 pg. With this assay method, the amounts of PTTH in the central nervous system and hemolymph were quantified. PTTH was detected only in the brain within the central nervous system, and, in the fifth instar, its content in the brain increased gradually with larval growth and decreased rapidly after the beginning of wandering. A substantial amount of PTTH was also found in the retrocerebral complex of day-3 fifth instar larvae, accounting for 28% of total PTTH. The PTTH titer in hemolymph changed dramatically during Bombyx development, with a small peak in the middle of the fourth instar, medium-sized peaks at the wandering and prepupal stages in the fifth instar, and a large prolonged peak during early pupal-adult development. The changes were overall closely correlated with those in hemolymph ecdysteroid titer. However, some unexpected aspects of PTTH dynamics in hemolymph have also been disclosed. Based on these observations, the significance of PTTH secretion in the control of insect development is discussed.     

Monoclonal antibody to shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity

A monoclonal antibody, 5-5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed. An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue. This result and our previous results using an anti-Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.     

A novel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ,SJL/J,and SWR/J mice

Idiopathic dilated cardiomyopathy (DCM) is responsible for approximately 25% of all cases of congestive heart failure. We have recently shown that immunization of autoimmune-susceptible SWXJ mice with whole cardiac myosin leads to T cell-mediated experimental autoimmune myocarditis (EAMC) and DCM. We have now identified two disease-inducing peptides from cardiac alpha-myosin heavy chain (CAMHC). Our approach involved the use of a novel MHC class II-binding motif contained in several peptides known to be immunogenic in SWXJ (H-2(q,s)) mice or in the parental SJL/J (H-2(s)) or SWR/J (H-2(q)) mouse strains. Two of four CAMHC peptides containing the -KXXS- peptide motif were found to be immunogenic. Immunization of SWXJ or parental SJL/J and SWR/J mice with CAMHC peptides palpha406-425 or palpha1631-1650 resulted in EAMC and DCM, characterized by inflammation, fibrosis, and decompensated right-sided ventricular dilatation. Despite mediating high incidences of severe disease, both peptides were found to be cryptic determinants, thereby providing further evidence for the importance and perhaps predominance of self crypticity in autoimmunity. Both peptides showed dual parental I-A(q) and I-A(s) restriction and mediated passive transfer of disease with activated CD4(+) T cells. An intact motif was necessary for antigenicity because loss of activity occurred in peptides containing nonconservative substitutions at the motif's terminal lysine and serine residues. Our studies provide a new model for EAMC and DCM in strains of mice widely used in autoimmune studies. Moreover, the -KXXS- motif may be particularly useful in implicating previously overlooked proteins as autoimmune targets and in facilitating the development of new organ-specific autoimmune mouse models for human diseases.     

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.     

Dual abnormal effects of mutant MITF encoded by Mi(wh) allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation

Structural localization of a peptide region, KRQPRNPKTDKLVNE, in the catalytic subunit of (Na(+) + K(+))-ATPase was investigated using a specific antibody directed against this peptide in cultured African green monkey kidney CV-1 cells. Immunofluorescence staining of frozen cell sections shows that an anti-KRQPRNPKTDKLVNE antibody (SSA95) interacts with its antigenic site and binds to the extracellular side of the cell membrane. Indirect immunofluorescence and flow cytometric analyses confirmed the presence of this epitope on intact cell surfaces. These results suggest that the KRQPRNPKTDKLVNE region of the (Na(+) + K(+))-ATPase is expressed on the cellular membrane surface.     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

Discriminative determination of alkyl methylphosphonates and methylphosphonate in blood plasma and urine by gas chromatography-mass spectrometry after tert.-butyldimethylsilylation

A method for determining two nerve gas hydrolysis products, alkyl (ethyl, isopropyl and pinacolyl) methylphosphonates (RMPAs) and methylphosphonate (MPA), separately, in human plasma and urine samples was developed, using two different deproteinization procedures. In the first method, the plasma sample was deproteinized by adding a fourfold volume of acetonitrile, followed by passing the supernatant through a Bond Elut strong anion-exchange (SAX) cartridge [fluoride (F(-)) form]. After washing the cartridge with water and methanol, the RMPAs were eluted with a 3% (v/v) solution of methanolic ammonia, and analyzed by gas chromatography-mass spectrometry (GC-MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. The detection yields of TBDMS derivatives of RMPAs were in the range of 69 to 99%, in contrast to the poor yields obtained when only acetonitrile deproteinization pretreatment was used (yield: 13-26%). The yield of the TBDMS derivative of MPA was very low (8%), however. In a the second method, a plasma sample was deproteinized by adding a half volume of 10% (w/v) trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA, the aqueous fraction was then passed through a Bond Elut SAX cartridge. After washing the cartridge with 0.5% (v/v) methanolic ammonia, MPA was eluted with 3% (v/v) methanolic ammonia. The detection yield of the TBDMS derivative of MPA was nearly quantitative. A pretreatment method using SAX solid-phase extraction was also developed for the cleanup of a urine sample, in which the sample was directly applied to a Bond Elut SAX cartridge, followed by elution of the RMPAs and MPA with 3% (v/v) methanolic ammonia, which were then derivatized and analyzed by GC-MS. The detection yields of TBDMS derivatives of RMPAs and MPA were in the range of 61 to 97%.