Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models.     

Abnormal accumulation of galactosylceramide in the kidney of twitcher mouse

The kidney tissue of the twitcher mice, a neurological mutant caused by a genetic deficiency of galactosylceramidase, contains enormously increased amounts, up to 50 times normal, of galactosylceramide. The finding is in sharp contrast with those in the enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease), in which no specific abnormal accumulation of galactosylceramide occurs despite the same genetic block in the catabolic pathway. This indicates that the same genetic defect can result in entirely different consequences in different species. Caution must be exercised even when "authentic animal models" are utilized for studies of human diseases.     

Histochemical demonstration of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues by means of almond glycopeptidase digestion

Almond glycopeptidase is an enzyme which cleaves specifically beta-aspartylglucosylamine linkages in glycoproteins with asialo-carbohydrate moieties. With this enzyme, it was possible to demonstrate the localization of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues. In these tissues, the oligosaccharides were shown to react positively for a series of histochemical procedures for neutral complex carbohydrates such as periodic acid-Schiff (PAS), peroxidase-labelled Ricinus communis agglutinin-I-diaminobenzidine (PO-RCA-DAB) and concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB). The asparagine-linked carbohydrates were localized in the placental villi, blood vessels and perivascular tissues and the umbilical cord blood vessels and matrix. The results of previous biochemical analyses performed upon the same tissues (Takahashi et al., 1981) have corroborated the results of the histochemical studies. The present results appear to substantiate the usefulness of almond glycopeptidase for the histochemical demonstration of the particular oligosaccharides of glycoproteins in tissues in general.     

Intergenus cell fusion between L-form cells of Pseudomonas aeruginosa and Escherichia coli

Intergenus cell fusion of prokaryotic bacteria was demonstrated for the first time; namely, fusion products doubly resistant to streptomycin and tetracycline were produced by polyethylene glycol treatment of a mixture of the streptomycin-resistant L-form of Pseudomonas aeruginosa and tetracycline-resistant L-form of Escherichia coli.     

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.     

Ferredoxins in Developing Spinach Cotyledons: The Presence of Two Molecular Species

An antibody for ferredoxin was used to investigate the developmentof ferredoxin during the greening of spinach cotyledons. Ferredoxinwas present in 8-day-old etiolated cotyledons and increasedwith illumination, which means that the synthesis of ferredoxinwas both light dependent and independent. The ferredoxin purified from etiolated cotyledons, greeningcotyledons, and mature leaves was a mixture of two chemicallydistinct molecular species; ferredoxin I and II. The relativecontents of these two species varied with the stage of developmentand the conditions used. Ferredoxin I was identical with that isolated previously asvalidated by its amino acid sequence [Matsubara and Sasaki (1968)J. Biol. Chem. 243: 1732]. The complete amino acid sequenceof the second component, ferredoxin II, was determined as well.It was composed of 97 amino acid residues and differed fromferredoxin I by 25 residues. (Received October 16, 1982; Accepted December 14, 1982)     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)     

Suppression of the responsiveness of lymphocytes from cancer patients triggered by co-culture with autologous tumor-derived cells

The question as to whether or not cancer patients have "tumor antigen"-induced suppressor T cells is of considerable interest and importance. As an approach to that question, the effect of addition of autologous irradiated tumor-derived cells (TDC) on the mixed lymphocyte response (MLR) of patients' lymphocytes (Ly) and of healthy donor Ly was tested. The rationale for these experiments was based on the fact that circulating antigen-responsive blood lymphocytes can be reactivated in vitro by exposure to the appropriate antigen. Thus, if there are circulating tumor "antigen"-reactive suppressor Ly, exposure to TDC as a source of the antigen should reactivate those cells. Reactivation of suppressor cells might result in diminished responsiveness to other stimuli such as alloantigens in the mixed leukocyte culture. We found that the addition of TDC to Ly cultures produced four distinct patterns of reaction. In 26 of the 74 different patient-tumor assays, the addition of autologous TDC to the patient cultures inhibited MLR, but the addition of the same TDC to cultures of Ly from healthy donors had no effect or increased their responsiveness (Specific Suppression). In 21 cases, the addition of autologous TDC to the patient cultures suppressed the MLR and the addition of the same TDC to control cultures suppressed the response of some but not all the healthy donors (Selective Suppression). In four cases, the addition of TDC to the cultures suppressed the MLR of the patients and all of the control donors (Nonspecific Suppression). In 23 cases, the addition of autologous TDC resulted in no suppression of the patient MLR or of any of the simultaneously tested normal donors (No Suppression). When TDC of patients with noninvasive bladder cancer were added to their own Ly cultures, only four of 11 produced specific or selective suppression compared to 11 of 12 when TDC came from patients with superficially invasive cancer. These data provide indirect evidence to support the hypothesis that human tumors induce circulating suppressor cells that may be reactivated in vitro by co-culture with TDC.