Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible Iines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35^S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.     

Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

A vegetative storage protein （VSP） with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions： nitrogen storage and defense.     

Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

Monochamus alternatus Hope （Coleoptera： Cerambycidae） is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides （organophosphates and pyrethroids）, which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro 32P-labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate （cAMP）, deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin （Ca^2＋CaM）-stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.     

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(CT) or A→G(AG) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F_0 are mosaic, and breeding of F_1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F_0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only CT mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F_0 male embryos are homozygous and three of them were Dmd precise CT mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a CT mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F_0 embryos with maternal point mutated alleles in a single step.     

Evidence of somatic embryogenesis from root tip explants of the rattan Calamus manan

Summary Somatic embryogenesis of Calamus manan, a single-stemmed rattan species, in tissue culture was scientifically demonstrated for the first time. Root tips of in vitro plantlets produced friable callus when the explants were cultivated for several mo. on a Murashige and Skoog induction medium containing 7.5 mg Picloram per l (31.1 μM). Histological analyses established the presence of proembryos within the callus which differentiated subsequently into somatic embryos using the same culture medium. Histological examination revealed that these somatic embryos completely lacked starch and protein reserves, which did not prevent them, however, from germinating, and showing bipolar development. These somatic embryos further developed into young plants, similarly to zygotic embryos.     

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.     

Disruption effect of Microplitis bicoloratus polydnavirus EGF-like protein, MbCRP, on actin cytoskeleton in lepidopteran insect hemocytes

Microplitis bicoloratus is a braconid endoparasitic wasp associated with the polydnavirus named Microplitis bicoloratus bracovirus （MbBV）. Parasitism of Spodoptera litura larvae leads to an impaired cellular immune response and to the disappearance of the 42 kDa actin in host hemocytes. In this work, we investigated if the absence of actin in blood cells was related to MbBV infection. An MbBV gene similar to egf-like genes identified in another bracovirus was partially cloned and named Mbcrp1. The full-length gene, named Mbcrp, is transcribed throughout the course of parasitism in host hemocytes and the 30 kDa MbCRP protein was detected in hemocytes 6-7 d post-parasitization. The Mbcrp1 gene contains the cysteine-rich trypsin inhibitor-like （TIL） domain coding sequence and the expression of recombinant MbCRP1 inhibited the expression of the 42 kDa actin in Hi5 cells. The 34.1 kDa MbCRPl-green fluorescent protein fusion protein locate specifically in the cytoplasm. These results suggest that expression of MbCRP in lepidopteran insect cells is related to the disruption of the actin cytoskeleton.     

丙型肝炎病毒感染的体外培养细胞的抗原表达

建立丙型肝炎病毒（HCV）体外感染细胞模型,观察其感染细胞的HCV抗原表达,用抗HCV抗体和HCVRNA阳性及阴性血清感染MOLT-4细胞,制备细胞片进行免疫酶染色。结果显示HCV感染MOLT-4细胞4天和阴性对照HCV抗原均为阴性;感染后7天胞浆内可见HCV抗原阳性免疫反应产物;15天阳性细胞达到高峰,43天仍可见少量阳性细胞。结果表明HCV体外感染MOLT-4细胞胞浆内观察到HCV抗原表达。     

Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates.

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

Substrates of ADP-ribosyltransferase of human erythrocytes

Incubation of human erythrocyte membranes with [32P]NAD resulted in the label incorporation into the 37 kDa and 41 kDa proteins as determined by SDS-PAAG electrophoresis with subsequent autoradiography. Treatment of labeled membranes with HgCl2 caused a significant decrease of the protein band intensity in the autoradiograms. Incubation of purified GTP-binding protein from rat brain (Go-protein) with membranes and erythrocyte cytoplasm in the presence of [32P]NAD resulted in the label incorporation into the Go-protein alpha-subunit. This incorporation was markedly decreased after treatment of radiolabeled Go-protein with HgCl2. The data obtained testify to the existence in human erythrocytes of cytoplasmic and membrane-bound forms of cysteine-specific ADP-ribosyl transferase, for which the Go-protein serves as a substrate. The 37 kDa and 41 kDa proteins are substrates for the membrane-bound form of the enzyme.     

Purification and characterization of a 32-kDa protein that localizes to the sea urchin extraembryonic matrix, the hyaline layer.

We have purified a 32 kilodalton (kDa) protein that localized with isolated, intact hyaline layers prepared from 1-h-old embryos. The protein appeared not to bind calcium and was not quantitatively released from 1-h-old embryos in the absence of Ca2+ and Mg2+. Using polyclonal antiserum prepared against the 32-kDa protein, the antigen was detected throughout embryonic development. By the hatched blastula stage of development, the 32-kDa protein was replaced by a species of slightly smaller molecular mass. Quantitative determination indicated that the 32-kDa protein accounted for approximately 6% of the total protein present in the sea urchin egg. This result is suggestive of a structural role for the 32-kDa protein that is required throughout embryonic development, although perhaps in a modified form from the hatched blastula stage on.     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.     

In vitro and in vivo association of an oviduct estrus-associated protein with bovine zona pellucida

The interaction between the bovine egg zona pellucida and a 97 kDa estrus-associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus-derived oviduct fluid was more intense than was ampulla-derived oviduct fluid collected near estrus. The zonae pellucidae of 7-day-old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri-estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus-associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.