Isolation and Characterization of Factors in Sweet Potato Root Which Agglutinate Germinated Spores of Ceratocystis fimbriata, Black Rot Fungus

A factor which agglutinates the germinated spores of Ceratocystis fimbriata was isolated from the sweet potato root. The factor is a glycoprotein with a molecular weight of 1.6 × 10⁶ daltons and required divalent cations such as Ca²⁺, Mn²⁺, Ni²⁺, and Mg²⁺ for activity. The activity of the factor was pH-dependent. The factor also agglutinated rabbit erythrocytes and is classified as a phytohemagglutinin or lectin. The factor agglutinated germinated spores of seven strains of C. fimbriata to almost the same degree. The factor showed differential agglutinating activity toward the strains in the presence of unidentified low molecular weight factor(s) in the sweet potato root. These results support our earlier suggestion that the spore-agglutinating factors in host plants function as the determinants of specificity in some host-parasite interactions.     

Immune Response to Toxoplasma gondii

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [³H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Transmembrane signal transduction and osmoregulation in Escherichia coli. Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ

The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation. In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions. It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane. Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity. It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected. These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus.     

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.     

Spot determinations of urinary cortisol for the screening of Cushing's syndrome.

The usefulness of spot determination of urinary cortisol in the screening of Cushing's syndrome was evaluated by measuring the cortisol concentration in randomly sampled urine in 68 normal subjects and in 9 patients with Cushing's syndrome. The urinary cortisol concentration in the morning was significantly higher in patients with Cushing's syndrome but some overlap existed between normal subjects and patients with Cushing's syndrome. In contrast, there was a clear discrimination between two groups when urinary cortisol was measured in the late evening: urinary cortisol was lower than 75 micrograms per gram creatinine (microgram/gCr) in normal subjects but higher than 150 micrograms/gCr in patients with Cushing's syndrome. When 1 mg dexamethasone was administered at 2300 h in the evening, spot urinary cortisol the next morning was less than 80 micrograms/gCr in normal subjects while it was above 100 micrograms/gCr in patients with Cushing's syndrome. Dexamethasone-induced suppression of urinary cortisol in normal subjects lasted until late in the afternoon, which allows sampling of urine at any time in the morning and possibly in the afternoon. These results suggest the usefulness of spot determination of urinary cortisol in the screening of Cushings' syndrome.     

Classification and ecological characterization of coniferous forest phytogeocoenoses of Hokkaido,Japan

Coniferous forest phytogeocoenoses of Hokkaido Island, Japan, were studied to classify them based on vegetation characteristics, to analyse their soils, to correlate the vegetation and soil characteristics, and to provide some ecological interpretation for the phytogeocoenosis differentiation and establishment. Five forest types were distinguished based on the vegetation structure, each of which was comparable to plant association of Krajina (1960); 1. the moss type, 2. the Sasa kurilensis type, 3. the Sasa senanensis type, 4. the Carex sachalinensis type, and 5. the Dryopteris crassirhizoma type. Soil base status indicated by pH, electric conductivity, amount of calcium and magnesium, and base saturation showed a fair correlation with the forest types. The forest types were, therefore, arranged along a soil nutritional gradient. The moss type developed in the least fertile habitats whereas the Dryopteris crassirhizoma type in the most fertile habitats, and others were in between the two. It was suggested that in the island, where climate was humid with excess of soil water throughout a year, soil nutritional regime, more specifically availability of bivalent cations which tended to be removed by the excessive soil water, seemed to be a critical factor to differentiate and establish the forest types.This study was financially supported by the Science Research Grant of the (Monbusho) Japanese Goverment (Grant No. 60540422).     

Electrophoresis of mutant proteins in inherited diseases

The electrophoretic approaches for detection of mutant proteins in inherited diseases are briefly reviewed and discussed. Mutation of a protein, known to be associated with a specific inherited disease, is detected by immunoblotting, immunoprecipitation or enzyme staining, combined with various electrophoretic techniques. Some instrumental and technological devices for two-dimensional electrophoresis have been reported for the screening of mutant proteins in diseases of currently unknown etiology.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.