Proteins and Carbohydrates in Xylem Sap from Squash Root

The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml^–1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 48–64 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992)     

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Summary Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10^–4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation. Correspondence to: K. Sode     

Double-Blind Study of the Chronopharmacotherapy of Depression

To investigate the possibility of chronotherapy with antidepressants for patients with depression, we gave single daily doses of clomipramine 150 mg/day to 30 patients with depression at three different times of day, i.e., morning, noon, or before bedtime, using a double-blind method over a 4-week period. Beneficial effects differed according to the administration time of day, with the most effective result being found for the administration at noon. Time-dependent differences in side effects were observed in tremors and dryness of mouth. An additional 10 patients were administered their medications three times a day by traditional, equally divided doses, and the efficacy was inferior than daily single doses at noon. The study results showed the significance of the administration time of day for the benefits and side effects of antidepressant therapy for depression.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Effect of fatigue on maximal inspiratory pressure-flow capacity.

The inspiratory muscles can be fatigued by repetitive contractions characterized by high force (inspiratory resistive loads) or high velocities of shortening (hyperpnea). The effects of fatigue induced by inspiratory resistive loaded breathing (pressure tasks) or by eucapnic hyperpnea (flow tasks) on maximal inspiratory pressure-flow capacity and rib cage and diaphragm strength were examined in five healthy adult subjects. Tasks consisted of sustaining an assigned breathing frequency, duty cycle, and either a "pressure-time product" of esophageal pressure (for the pressure tasks) or peak inspiratory flow rate (for the flow tasks). Esophageal pressure was measured during maximal inspiratory efforts against a closed glottis (Pesmax), maximal transdiaphragmatic pressure was measured during open-glottis expulsive maneuvers (Pdimax), and maximal inspiratory flow (VImax) was measured during maximal inspiratory efforts with no added external resistance before and after fatiguing pressure and flow tasks. The reduction in Pesmax) with pressure fatigue (-25 +/- 7%) was significantly greater than the change in Pesmax with flow fatigue (-8 +/- 8%, P less than 0.01). In contrast, the reductions in Pdimax (-11 +/- 8%) and VImax (-16 +/- 3%) with flow fatigue were greater than the changes in Pdimax (-0.6 +/- 4%, P less than 0.05) or VImax (-3 +/- 4%, P less than 0.05) with pressure fatigue. We conclude that respiratory muscle performance is dependent not only on the presence of fatigue but whether fatigue was induced by pressure tasks or flow tasks. The specific impairment of Pesmax and not of Pdimax or flow with pressure fatigue may reflect selective fatigue of the rib cage muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

A small GTP-binding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol

We have recently purified from bovine brain cytosol a novel type of regulatory protein for smg p25A, named smg p25A GDP dissociation inhibitor (GDI), that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. This smg p25A GDI is inactive for other ras p21/ras p21-like small GTP-binding proteins (G proteins) including c-Ha-ras p21, smg p21, rhoA p21 and rhoB p20. In human platelet membranes, smg p25A was not detected but a G protein with an apparent Mr value of 24,000 (24KG) was recognized by smg p25A GDI and the dissociation of GDP from and the binding of GTP to 24KG were inhibited by smg p25A GDI. The doses of smg p25A GDI necessary for these activities for both 24KG and smg p25A were the same. This 24KG was not recognized by an anti-smg p25A monoclonal antibody. The GDI activity for human platelet 24KG and smg p25A was detected in human platelet cytosol. This human platelet GDI was recognized by an anti-smg p25A GDI polyclonal antibody. These results indicate that there is a 24KG-24KG GDI system similar to a smg p25A-smg p25A GDI system in human platelets.