Carbonic anhydorase isoenzyme I (CA-I) concentration in feces and urine as a temporary marker of occult blood in beagle dogs.

This study was undertaken to investigate whether the concentration of carbonic anhydorase isoenzyme I (CA-I) in canine feces and urine is useful as a temporary marker of occult blood. Concentrations of CA-I were measured by enzyme-linked immunosorbent assay (ELISA). Fecal CA-I concentrations in 113 healthy beagle dogs (50 male and 63 female) of various ages ranged from 4.3 to 16.7 ng/g feces (mean; 7.0 +/- 2.9 ng/g feces). One milliliter of blood from 3 healthy beagle dogs was found to contain 1,047, 1,062 and 1,150 microg CA-I. The fecal CA-I concentrations of dogs receiving intragastric infusions of autologous blood (10 ml) were very low. However, the fecal CA-I concentrations of dogs receiving infusion of autologous blood (5 ml) into the ascending colon were very high. Detection of fecal CA-I would be useful for identifying dogs with hemorrhaging of the large intestine. Of 55 urinary samples collected from healthy beagle dogs by catheter, chemical tests for occult blood were negative in 44, but CA-I concentrations ranged from 1.8 to 12.6 ng/ml (mean; 6.9 +/- 5.4 ng/ml) by ELISA. The CA-I concentrations of the other 11 samples, which tested positive for occult blood on chemical testing, ranged from 41.2 to 525.0 ng/ml by ELISA. Although CA-I is not a specific marker of erythrocytes, CA-I may be used to detect occult blood in canine feces and urine until a specific immunological test kit using antibody for Hb is developed.     

Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.     

Molecular analyses of glucosyltransferase genes among strains of Streptococcus mutans

Three glucosyltransferase (GTase) genes (gtfB, gtfC and gtfD) were cloned and sequenced from clinically isolated strains of Streptococcus mutans MT8148 (serotype c), MT4239 (c), MT4245 (e), MT4467 (e) and MT4251 (f), respectively. Comparison of the gtf genes revealed that interstrain difference of gtfB and gtfD was limited, while gtfC showed significant interstrain variations. Similar to gtfB and gtfD, gtfC possessed five direct repeats composed of homologous unit in the carboxyl-terminal portion. The repeating unit consisted of 63–65 amino acid residues and is responsible for glucan binding. The gtfC gene from S. mutans MT4245 lacked the fourth unit. Multiple alignment with the gtf sequence of strain GS-5 (c) revealed several changes in these gtf genes due to frameshift mutations. The peptides encoded by the gtfB, gtfC and gtfD genes of GS-5 were 1, 80, and 32 amino acid residues shorter than those of the test strains except strain MT4245.     

Enantioselectivity of bunitrolol 4‐hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450‐2D6

The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc.     

Innate immune response to viral infection

In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.     

The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase

A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.     

Synthesis of 1-arylpiperazyl-2-phenylcyclopropanes designed as antidopaminergic agents: cyclopropane-based conformationally restricted analogs of haloperidol

A series of the cyclopropane-based conformationally restricted analogs of haloperidol were designed as potential antidopaminergic agents and were effectively synthesized using highly stereoselective Grignard reaction with tert-butanesulfinyl imines as the key step. Pharmacological evaluation of the compounds showed that the conformational restriction method can effectively work for improving the pharmacological selectivity of a parent compound and also for investigating the bioactive conformation.     

RNA-binding protein hoip accelerates polyQ-induced neurodegeneration in Drosophila

Abnormal polyglutamine (polyQ) expansion in the N-terminal domain of the human androgen receptor (hAR) is known to cause spinobulbar muscular atrophy (SBMA), a hereditary human neurodegenerative disorder. To explore the molecular mechanisms of neurodegeneration in SBMA, we genetically screened modulators of neurodegeneration in a Drosophila SBMA experimental model system. We identified hoip as an accelerator of polyQ-induced neurodegeneration. We found that hoip forms a complex with 18s rRNA together nop56 and nop5 proteins, whose human homologs are known to form a snoRNP complex involved in ribosomal RNA processing. Significantly, the levels of mutant polyQ-hAR were up-regulated in a mutant line overexpressing hoip. Consistently, severe neurodegeneration phenotype (rough eye) was also observed in both nop56 and nop5 overexpression mutant lines. These findings suggest that the process of neurodegeneration induced by abnormal polyQ expansion in the hAR may be regulated by the activity of snoRNP complex.