Protein-tyrosine phosphorylations induced by concanavalin A and N-formyl-methionyl-leucyl-phenylalanine in human neutrophils.

The ability of the lectin concanavalin A (ConA) and N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce protein-tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80-kDa and 40-kDa proteins. These protein-tyrosine phosphorylations were time- and dose-dependent. The tyrosine phosphorylation of 40-kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40-kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule-associated protein 2 (MAP2) kinase suggested that this tyrosine-phosphorylated 40-kDa protein might be the MAP2 kinase.     

Isolation and identification of lepidimoide, a new allelopathic substance from mucilage of germinated cress seeds

A new allelopathic substance that promoted the shoot growth of different plant species but inhibited the root growth was isolated as an amorphous powder from mucilage of germinated cress (Lepidium sativum L.) seeds. This substance was identified as sodium 2-O-rhamnopyranosyl-4-deoxy-threo-hex-4-enopyranosiduronate (designated lepidimoide) from the mass and the nuclear magnetic resonance and infrared spectra coupled with some chemical evidence. Lepidimoide promoted the hypocotyl growth of etiolated Amaranthus caudatus L. at concentrations higher than 3 μm and inhibited the root growth at concentrations higher than 100 μm. The growth-promoting activity in hypocotyls was 20 or 30 times as much as that of gibberellic acid.     

α-1-Antichymotrypsin variant detected by PCR-single strand conformation polymorphism (PCR-SSCP) and direct sequencing

A new mutant -1-antichymotrypsin (variant ACT) was found by polymerase chain reaction single strand conformation polymorphism and direct sequencing. In this variant ACT, two bases (AA) were deleted from codon 391. This resulted in a different amino acid sequence downstream of the deletion point, elongating the peptide chain by 10 amino acids.     

No severe bottleneck during human evolution: evidence from two apolipoprotein C-II deficiency alleles.

The DNA sequences of a Japanese and a Venezuelan apolipoprotein (apo) C-II deficiency allele, of a normal Japanese apo C-II gene, and of a chimpanzee apo C-II gene were amplified by PCR, and their nucleotide sequences were determined on multiple clones of the PCR products. The normal Japanese sequence is identical to--and the chimpanzee sequence differs by only three nucleotides from--a previously published normal Caucasian sequence. In contrast, the two human mutant sequences each differ from the normal apo C-II gene sequence by several nucleotides, including deletions. The data suggest that both mutant alleles arose greater than 500,000 years ago. It is shown that a defective allele can persist in a population for only a short time if a bottleneck occurs. Therefore, the antiquity of the two alleles suggests no severe bottleneck during human evolution. Moreover, the fact that one allele is from Japan and the other is from a Venezuelan Caucasian family is more consistent with the multiregional evolution model of modern human origins than with the complete replacement or "out of Africa" model.     

Delta opioid receptor-selective ligands: [D-Pen2,D-Pen5]enkephalin-dermenkephalin chimeric peptides.

A number of DPDPE-dermenkephalin chimeric peptides have been synthesized in which the putative C-terminal delta-address of dermenkephalin has been linked to the highly delta opioid selective cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE). Asp, Met-Asp and Leu-Met-Asp have been added to the C-terminus of DPDPE and both the carboxyl terminal and the carboxamide terminal series have been prepared. The bioassays using the mouse vas deferens and guinea pig ileum preparations have revealed a steady decrease in potency (compared to DPDPE) at delta and mu receptors as the dermenkephalin sequences were added. Some of the analogues, however, retained high delta selectivity. Similar results were obtained using radioligand binding assays. These findings suggest that the C-terminal amino acid sequence of dermenkephalin plays a role of delta-address which is specific to dermenkephalin itself, and is not additive with another delta selective ligand such as DPDPE.     

Topographical requirements for delta opioid ligands: presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity.

To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats.     

The lectin wheat germ agglutinin stimulates a protein-tyrosine kinase activity of p72syk in porcine splenocytes

We previously reported a molecular cloning of porcine gene syk encoding a non-receptor type 72-kDa protein-tyrosine kinase and a generation of anti-CPTK40 antibodies which could immunoprecipitate the activity of p72syk (Taniguchi et al. (1991) J. Biol. Chem. 266, 15790-15796). In this study, we have demonstrated that wheat germ agglutinin caused an increase in the autophosphorylation activity of p72syk which preceded an increase of protein-tyrosine phosphorylation observed at the same time in porcine splenocyte. The increase of p72syk activity was dose dependent and was inhibited by the coexistence of N-acetyl-D-glucosamine. Upon stimulation by the combination of 4 beta-phorbol 12-myristate 13-acetate and ionophore A23187, we could not detect the increase of activity of p72syk suggesting that the activation of p72syk was independent of protein kinase C and calcium ions.     

Effect of a glucan,sizofiran, on natural-killer activity of 5-fluorouracil-treated murine bone marrow cells

Summary The number of bone marrow cells in C3H/He mice was reduced 3–4 days after treatment with 130 mg/kg intraperitoneal 5-fluorouracil (5-FU). Higher rates of spontaneous proliferation and natural killer (NK) activity, accompanied by an increase in asialoGM1-positive cells, were observed in treated mice. When sizofiran at a dose of 200 µg/animal was intramuscularly injected after 5-FU treatment, the rates of proliferation and NK activity of bone marrow cells were higher than with 5-FU alone. The cell number was not influenced by sizofiran alone. These results indicate that all precursors of the various mature cell types (including NK cells) differentiate and regenerate rapidly to replace cells damaged by 5-FU treatment, and that sizofiran has the potential to assist this recovery. These results suggest that administration of sizofiran after chemotherapy may be useful in cancer patients.     

Assay for the (R)- and (S)-enantiomers of salsolinols in biological samples and foods with ion-pair high-performance liquid chromatography using β-cyclodextrin as a chiral mobile phase additive

A chromatographic procedure was devised for the quantitative determination of the enantiomers of salsolinol and N-methylsalsolinol, which are biologically important alkaloids. The enantiomers of salsolinol and N-methylsalsolinol were completely separated using β-cyclodextrin in a reversed-phase ion-pair system. The HPLC method was sensitive enough to detect the isoquinolines at a concentration less than 0.1 pmol per injection. The presence of (R)- and (S)-salsolinol was confirmed in fermented foods and beverages, while N-methylsalsolinol was not detected. On the other hand, the (R)-enantiomers of both salsolinol and N-methylsalsolinol were found to predominate in the human brain.     

Co-existence of subadult males and females as alternative tactics of breeding post acquisition in a monogamous and protandrous anemonefish

Summary The monogamous and protandrous anemonefish,Amphiprion clarkii, utilizes only large host sea anemones as breeding sites. Under conditions of breeding site shortage, an important strategy for unmated fish may be to prepare themselves to enter quickly a breeding site from which a breeding male or female has recently disappeared. There are two types of subadults. Some juveniles become subadult males, which can change either to male or female afterwards, but others become subadult females, which can change only to female. Subadult females seem to acquire female posts more easily than subadult males. We develop an ESS model which evaluates the future reproductive success of subadult females and subadult males and test whether coexistence of subadult males and females can be interpreted as adaptive alternative tactics, using field data from two populations. The model predicts that if mortality does not differ between adult males and females the frequency for juveniles to become subadult females should be equal to the proportion of breeding post acquisition by subadult females among that by all subadults. The model also predicts that if mortality does not differ between subadult males and females, nor in adults, the frequency of subadult females among all subadults should be equal to the proportion of post acquisition. The model, with data from two study sites, suggests that alternative life-history pathways found inA. clarkii have evolved through adaptive tactics of breeding post acquisition.