Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

火炬松体细胞无性系建立及其形态发生调控的研究

火炬松（Pinus taeda L.）原产美国东南部，是世界南方松中最重要的绿化和造林用速生针叶树种，现广泛分布于全球亚热带和部分热带地区，在我国的栽植面积居世界第2位，仅次于美国。目前，火炬松离体快速繁殖、遗传转化和品种改良研究中最大的障碍是没有获得良好的植株再生体系。本研究以火炬松的成熟种子为试材，建立了可调控的体细胞胚胎发生和器官发生植株再生系统，并对再生过程中的形态学变化进行了细胞学观察和扫描电镜观察;建立了胚性细胞悬浮系，测定了其重要生长参数的变化动态，优化了悬浮条件下体细胞胚胎发生的培养条件及悬浮细胞原生质体直接体细胞胚胎发生的培养条件。 进行了HA、HB、HC、MA、MB、MC、LA和LB等8种不同基因型的成熟合子胚在BMS、DCR、GD、LM、LP、MNCI、MS、SH及自行设计的TE等9种不同基本培养基上的愈伤组织诱导试验，筛选获得了愈伤组织发生频率较高的基因型HB、MA和MC，及基本培养基DCR和TE。激素组合试验表明，2,4-D和BA最有利于愈伤组织发生。两因子5水平等重复的愈伤组织诱导试验及方差分析结果证实，8 mg•L~(-1)2,4-D和4 mg•L~(-1)BA是愈伤组织诱导的最佳激素组合。诱导产生的愈伤组织经2次继代后，可明显分为4种类型，它们是1）白色、半透明、有光泽的粘性愈伤组织（WTGM）;2）淡黄色、疏松、有光泽的颗粒状愈伤组织（YLGG）;3）淡绿色、疏松的颗粒状愈伤组织（GLG）;和4）浅白色、水浸状的粘性愈伤组织（WMM）。其中白色、半透明、有光泽的粘性愈伤组织有较强的体细胞胚胎发生能力，淡黄色、疏松、有光泽的颗粒状愈伤组织有较强的不定芽发生能力。这两种愈伤组织的最高诱导频率分别是28.1%和35.7%。 在附加2,4-D、IBA和BA的DCR体细胞胚诱导培养基上，白色、半透明、有光泽的粘性愈伤组织中的胚性细胞形成胚性胚柄细胞团和早期原胚。提高培养基中的渗透压后，早期原胚发育成后期原胚。在附加ABA、PEG和活性炭的DCR体细胞胚成熟培养基上，后期原胚发育成子叶胚。在无激素DCR培养基上，子叶胚萌发形成再生完整植株。体细胞胚转换成小植株的最高频率是18.4%。在直接体细胞胚诱导增养基和直接体细胞胚发育培养基的作用下，成熟合子胚的子叶和胚轴上直接形成体细胞胚。直接体细胞胚胎发生的最高频率是18%。 在附加NAA、IBA和BA的TE不定芽原基诱导培养基上，淡黄色、疏松、有光泽的颗粒状愈伤组织中的胚性细胞形成不定芽原基。在附加IBA和BA的TE不定芽分化培养基上，不定芽原基分化产生不定芽。用基因型HB、MA和MC的淡黄色、疏松、有光泽的颗粒状愈伤组织进行的试验表明，不定芽分化的最佳低温（4 ℃）处理时间是5～6周，最佳蔗糖浓度是25～30 g•L~(-1)。分化产生的不定芽在附加IBA、GA_3和活性炭的TE培养基上，幼茎伸长。附加IBA、BA和GA_3的TE培养基上，伸长的不定芽生根形成完整植株。伸长不定芽的最高生根频率是46%。成熟合子胚在直接不定芽原基诱导培养基及直接不定芽分化培养基的作用下，从子叶和胚轴的不同部位产生直接不定芽。直接不定芽发生的最高频率是58.2%。 由成熟合子胚诱导愈伤组织形成过程中的形态学观察表明：在附加2,4-D和BA的DCR愈伤组织诱导培养基上，HB、MA和MC3种基因型中，MA主要在下胚轴形成愈伤组织，MC主要在子叶和胚根形成愈伤组织，HB主要在胚根形成体积较小的愈伤组织。在附加NAA和BA的TE愈伤组织诱导培养基上，HB、MA和MC3种基因型中，MA在单个子叶的顶端形成生长较快的愈伤组织，MC的所有子叶都形成愈伤组织，HB在所有子叶的顶端形成愈伤组织。 石蜡切片观察表明：4类愈伤组织的细胞组成不同．第1类愈伤组织主要由核大、质浓、体积小的园形胚性细胞及核呈柱状或新月形的体积较大的非胚性细胞组成;第2类愈伤组织主要由核大、质浓、体积小的园形胚性细胞组成，第3类愈伤组织主要由体积较大的棒状、葫芦形、新月形、盾片状细胞组成、第4类愈伤组织主要由体积较大的薄壁细胞和细胞壁加厚、细胞间连结紧密、无细胞核的分化细胞组成，第1类愈伤组织上形成的早期原胚的特点是：胚性头部由排列紧密、体积小的园形细胞组成，轮廓十分清晰、呈半圆形，胚柄由排列疏松的长形细胞组成，细胞体积大、细胞中有大的液泡，早期原胚在发育过程中和母体组织保持一定的隔离状态。第2类愈伤组织上形成的不定芽的特点是t结构上为单极性，其维管束和母体组织保持密切联系。扫描电镜观察表明;直接体细胞胚基部和母体组织保持较少的联系，其子叶是直立生长的．直接不定芽基部和母体组织保持较多的联系，其幼叶是向心卷曲生长的。 在培养周期内，基因型HB、MA和MC胚性细胞悬浮培养物的几个生长参数的变化动态相似。鲜重增长高峰在12—15 d，干重增长高峰在15～18 d，细胞体积增长高峰和胚数增长高峰在18—21 d。在培养的18—21 d，培养液中的pH值、电导率和蔗糖浓度接近或降到最低点。在悬浮培养条件下，体细胞胚形成的最佳起始细胞密度是5～6×103个／ml。继代培养时间延长，体细胞胚胎发生能力下降。热激处理促进基因型HB和MA体细胞胚的形成，抑制基因型MC体细胞胚的形成。在悬浮培养物中，观察到了裂生多胚。 对数生长期的火炬松胚性悬浮细胞，在以甘露醇作为渗透压稳定剂的酶混合液Cel-lulase“Onozuka”RS l%+Cellulase“Onozuka”R-10 2.5%+Pectolyase Y-23 0.2%的作用下，原生质体的产量和活力均最高。原生质体在DCR和KM8P两种培养基上形成了体细胞胚（包括胚性胚柄细胞团、早期原胚和后期原胚）．体细胞胚形成的最佳起始原生质体密度是7×l04个/ml，最佳ABA浓度是4 mg．L-I．

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

12 p.

The addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for advanced colorectal cancer: results of the MRC COIN trial.

Background: In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival.
Methods: In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448.
Findings: 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3—29·2] in the control group vs 17·0 months [9·4—30·1] in the cetuximab group; HR 1·04, 95% CI 0·87—1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0—12·5] in the control group vs 8·6 months [5·1—13·8] in the cetuximab group; HR 0·96, 0·82—1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5—27·4); KRAS mutant, 14·4 months (8·5—24·0); all wild-type, 20·1 months (11·5—31·7).
Interpretation: This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended.

Germ line Cysts and the Formation of the Germinal Epithelium During the Female Gonadal Morphogenesis in Cyprinus Carpio (Teleostei: Ostariophysi: Cypriniformes)

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

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line

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

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G1- or G2/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell “parasitism” (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337–5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss’s hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

Virus neutralization by germ-line vs. hypermutated antibodies

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Cκ) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in VH increased by 300-fold the binding strength of monovalent scFv-Cκ. The multivalent binding avidity of germ-line scFv-Cκ was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Cκ did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show “affinity maturation” effects of up to 1000-fold.

Somatic mosaicism in Wiskott–Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott–Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.