Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Intranuclear microinjection for transformation of tomato callus cells

An efficient method, called the culture plate method, was devised for microinjection of foreign materials into nuclei of tomato callus cells. The culture plate method, used in this study, is advantageous because cells suitable for microinjection can be selected microscopically and the injected cells subsequently cultured in the same plate. With this microinjection system, some foreign materials were injected into nuclei of callus cells without causing detrimental effects. Kanamycin-resistant callus clones were obtained 1 month after injection from single cells whose nuclei were microinjected with a NPT II DNA fragment of the pE2KX plasmid.     

Frequencies of Twelve Ascus-Types and Arrangement of Three Genes from Tetrad Data

Equations expressing the theoretical frequencies of twelve ascus-types in the tetrad analysis of a triply heterozygous diploid are described. Using these equations, a mapping procedure for a gene X, is proposed. The procedure requires that two genes, X and Y, of the same phenotype be heterozygous and that the map position of Y be known, and that another standard gene, Z, show an independent phenotype from X and Y. This procedure does not require the laborious allelism test of the segregants to determine the allelic 2:2 segregation in tetrads for the X and Y genes, which is indispensable for mapping by the conventional procedure. The exact placement of the X gene on a chromosome is possible by the chi2 minimization procedure in comparison with the expected frequencies of the six ascus-types or four spore-types deduced from the twelve expected ascus-types to give the optimal fit with the observed data.     

Induction of Haploid Callus from Isolated Microspores of Peony in vitro

The in vitro culture of isolated microspores of two cultivarsof Paeonia, P. lactiflora cv. "Kumoinotsuru" (2n=10) and cv."Toyonoakari" (2n = 10), was attempted. With both cultivars,some cultured microspores (3–6%) began to divide afterfive to seven days of culture. Those of "Kumoinotsuru" grewinto calli, whereas the others degenerated. Cytological examinationof the callus developed from a microspore revealed haploids,diploids or a mixture of both. No direct embryo formation frommicrospores was observed in this experiment. (Received December 1, 1980; Accepted January 16, 1981)     

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.     

Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.     

Prodigiosin-25 C

A water-insoluble red antibiotic pigment was isolated from mycelia of a strain of Streptomyces. It was found that the pigment is a new C₂₅-prodigiosin-analogue and the authors propose to designate it prodigiosin-25 C. The chemical structure (XI) has been deduced from visible absorption spectra, NMR spectra, mass spectra and analysis of degradation products of the pigment.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.