虾夷扇贝Patinopecten yessoensis (Jay)和仿刺参Apostichopus japonicus (Seienka)多倍体诱导研究

本文采用化学方法诱导了虾夷扇贝Patinopecten yessoensis (Jay)和仿刺参Apostichopus japonicus (Selenka)的多倍体。采用6－二甲基氨基嘌呤（6－dimethylaminopurine）抑制虾夷扇贝极体释放，研究了诱导三倍体的实验条件、技术工艺以及三倍体虾夷扇贝幼虫的生长和培育方法。结果表明，使用6－二甲基氨基嘌呤（6－DMAP）可诱导17.3%-100.0%的三倍体虾夷扇贝面盘幼虫。筛选并优化了生产中操作简便、高效、成活率高的诱导条件，并研究出在扇贝三倍体诱导中批量获得受精卵、处理及培育的技术工艺，对扇贝三倍体的规模化诱导及培育等生产性应用具有较高的参考价值。报道了采用细胞松弛素B（cytochalasin B）、秋水仙素（colchicine）、6－DMAP以及咖啡因（coffeine）等药物抑制虾夷扇贝第一极体（PB_1）释放、PB_1和第二极体（PB_2）释放以及抑制第一次卵裂等方法诱导四倍体的结果。结果表明，CB、6－DMAP和秋水仙素抑制扇贝第一次有丝分裂诱发四倍体的比例低于25%，而采用CB抑制PB_1可有效地诱导产生四倍体，从授精后42min提前到15-22min开始处理，抑制PB_1的放出有助于提高四倍体的比例，在12 ℃，处理开始和终上时间分别在授精后20－22min和60－62min时，面盘幼虫四倍体率最高，为56.5%。对CB处理抑制受精卵PB_1释放的处理组胚胎的染色体分离状况进行了观察研究。对照组受精卵具有19条四分体染色体，经过减数分裂I期（meiosis I）和减数分裂II期（meiosis II），放出PB_1和PB_2，受精卵的发育具有不同步性。处理组受精卵在第二次减数分裂中出现了“三级分离“、“联合二级分离“和“独立二级分离“等特殊的分离类型，初步分析了CB抑制第一极体放出的机理。对三倍体虾夷扇贝的繁殖潜力和卵子的大小进行了观察研究，三倍体扇贝具一定的繁殖能力，三倍体雌贝平均产卵3.26 * 10~6个，而相同壳长二倍体贝为1.45 * 10~7，三倍体产卵量仅为二倍体产卵量的22.5%。三倍体卵子产出后，形状不规则，卵子平均直径为87.25μm，比二倍体大11.7%，卵子体积比二倍体大39.4%。利用CB抑制第一极体释放诱导了虾夷扇贝的四倍体，诱导率为41.5%。首次报道了仿刺参Apostichopus japanicus (Selenka)多倍体诱导的结果。采用紫外线照射的海水成功地使海参分别单独产卵、排精，从而准确地控制了海参的人工授精后处理的时间。采用0.2、0.4mg/L CB 抑制受精卵第一极体释放以及10、20、30和40mg/L 的6－DMAP 分别抑制PB_1、PB_2放出的方法诱导了仿刺参的多倍体。研究了诱导的药物浓度、处理时间以及处理开始时间等，同时对幼体的成活率等进行了探讨。结果表明，2种药物均可诱导仿刺参产生三倍体和四倍体，从效果上看，采用CB抑制PB_1诱导，到达小耳幼体时，可产生9.7%-21.3%的四倍体。6－DMAP抑制PB_2放出诱导三倍体，三倍体诱导率介于7.5%-25.8%之间。

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

The effect of triclabendazole ("Fasinex") on protein synthesis by the liver fluke, Fasciola hepatica

The effect of the active sulphoxide metabolite of the anthelmintic triclabendazole (TCBZ-SX, 15-50 mu g ml(-1)) on the incorporation of radioactively labelled [C-14] leucine by adult Fasciola hepatica tissue slices was measured by liquid scintillation counting. In addition, the ability of the microfilament-disrupting drug, cytochalasin B, and the microtubule-disrupting drug, tubulozole-C, to inhibit protein synthesis, was assessed by similar methods and compared with TCBZ-SX. The established protein synthesis inhibitors, cycloheximide and actinomycin D were used as positive controls. All the drugs showed a significant inhibition of protein synthesis, albeit to different extents; however, TCBZ-SX was the most potent, with no significant difference between its effect and that of cycloheximide or actinomycin D. Moreover, the concentration of TCBZ-SX, above 15 mu g ml(-1), had little further influence on incorporation of [C-14] leucine. This investigation demonstrates the inhibitory effect of TCBZ-SX, cytochalasin B and tubulozole-C on protein synthesis in F. hepatica and confirms the qualitative observations made in several previous ultrastructural studies.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

Synapsin- and actin-dependent frequency enhancement in mouse hippocampal mossy fiber synapses

The synapsin proteins have different roles in excitatory and inhibitory synaptic terminals. We demonstrate a differential role between types of excitatory terminals. Structural and functional aspects of the hippocampal mossy fiber (MF) synapses were studied in wild-type (WT) mice and in synapsin double-knockout mice (DKO). A severe reduction in the number of synaptic vesicles situated more than 100 nm away from the presynaptic membrane active zone was found in the synapsin DKO animals. The ultrastructural level gave concomitant reduction in F-actin immunoreactivity observed at the periactive endocytic zone of the MF terminals. Frequency facilitation was normal in synapsin DKO mice at low firing rates (approximately 0.1 Hz) but was impaired at firing rates within the physiological range (approximately 2 Hz). Synapses made by associational/commissural fibers showed comparatively small frequency facilitation at the same frequencies. Synapsin-dependent facilitation in MF synapses of WT mice was attenuated by blocking F-actin polymerization with cytochalasin B in hippocampal slices. Synapsin III, selectively seen in MF synapses, is enriched specifically in the area adjacent to the synaptic cleft. This may underlie the ability of synapsin III to promote synaptic depression, contributing to the reduced frequency facilitation observed in the absence of synapsins I and II.

Substâncias antifúngicas de Xylaria sp., um fungo endofítico isolado de Palicourea marcgravii (Rubiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo químico e avaliação biológica dos fungos endofíticos Xylaria sp. e Colletotrichum crassipes isolados de Casearia sylvestris (Flacourtiaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

THE MICROCELL-MEDIATED TRANSFER OF SINGLE HUMAN CHROMOSOMES INTO RECIPIENT MOUSE CELLS

Microcell-mediated chromosome transfer is a method of gene transfer which allows for the introduction of single or small groups of intact chromosomes into recipient host cells. Microcell transfer was first performed by Fournier and Ruddle using rodent microcells and various recipient cells. Expansion of this technology to include the transfer of normal human genetic material has been hindered because large micronucleate populations from diploid human cells have been unobtainable. This dissertation research describes, however, the methods for production of micronuclei in 40-60% of normal human fibroblasts. Once micronucleate cells were obtained, they were enucleated by centrifugation in the presence of Cytochalasin B; the microcells were then purified and fused to recipient mouse (LMTK('-)) cells using a new fusion protocol employing polyethylene glycol containing phytohemagglutinin. Microcell clones were isolated from the HAT selection system. Alkaline Giemsa staining performed on these hybrids indicated the presence of a single human chromosome in each of seven microcell clones from three separate experiments. That chromosome was further identified by G banding analysis to be human chromosome #17, which codes for thymidine kinase. The time course for production of these hybrids from fusion to karyotypic analysis was 6 weeks. The viability of the transferred human genetic material was assessed by electrophoretic isozyme analysis.^ Subsequent experiments were performed in an attempt to optimize the transfer frequency for the thymidine kinase gene using this system. Results indicated that the frequency could be increased from < 1 x 10('-6) in initial experiments to 2 x 10('-5) in the latest experiment. Analyses were also conducted to determine the number of chromosomes per isolated microcell as well as to investigate the stability of the transferred human chromosome in the mouse genome. ^

Experiment: Growth rates and percentages of normal versus malformed versus incomplete versus incomplete and malformed coccoliths

The coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was cultured in natural seawater with the addition of either the microtubule-inhibitor colchicine, the actin-inhibitor cytochalasin B, or the photosynthesis inhibitor 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea (DCMU). Additionally, E. huxleyi was cultured at different light intensities and temperatures. Growth rate was monitored, and coccolith morphology analyzed. While every treatment affected growth rate, the percentage of malformed coccoliths increased with colchicine, cytochalasin B, and at higher than optimal temperature. These results represent the first experimental evidence for the role of microtubules and actin microfilaments in coccolith morphogenesis.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12°C). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos

Mouse clones were produced by serial nuclear transfer commencing with the transfer of four-cell nuclei at metaphase into unfertilized ooplasts. The donor four-cell-stage nuclei were synchronized in metaphase with nocodazole. The oocytes receiving a four-cell nucleus at metaphase formed two nuclei after artificial activation and inhibition of cytokinesis with cytochalasin B. To obtain embryos with diploid sets of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized one-cell embryos, thus doubling the number of identical embryos. This procedure produced a high frequency of development of reconstructed embryos to the blastocyst stage. Of 11 sets of identical embryos produced by serial nuclear transplantation, 83% developed into blastocysts, including three sets of identical septuplet blastocysts. After transfer to recipient mice, a total of 25 (57%) live young were obtained, which included one set of identical sextuplet and two sets of identical quadruplet mice.

A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.

Polymerizing Microtubules Activate Site-directed F-Actin Assembly in Nerve Growth Cones

We identify an actin-based protrusive structure in growth cones termed “intrapodium.” Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g., Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations.