Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L⁻¹ was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.

     

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Triage of Atypical Glandular Cell by SOX1 and POU4F3 Methylation: A Taiwanese Gynecologic Oncology Group (TGOG) Study

Introduction

Invasive procedures including loop electrosurgical excision, cervical conization, and endometrial sampling are often recommended when atypical glandular cells (AGC) are detected on Pap smear with unsatisfactory colposcopy. These invasive procedures may result in patient anxiety, increased medical expense, and increasing the risk of preterm delivery in subsequent pregnancies. This study was performed to assess methylation biomarkers in the triage of AGC on Pap smear for invasive procedures.

Methods

We conducted a multicenter study in 13 medical centers in Taiwan from May 2012 to May 2014. A total of 55 samples diagnosed “AGC not otherwise specified” (AGC-NOS) were included. All patients with AGC underwent colposcopy, cervical biopsy, endometrial sampling, and conization if indicated. Multiplex quantitative methylation-specific polymerase chain reaction (QMSPCR) was performed. Sensitivity, specificity, and accuracy were calculated for detecting CIN3⁺ and endometrial complex hyperplasia.

Results

In 55 patients with AGC, the sensitivity for methylated (^m) SOX1^m, PAX1 ^m, ZNF582^m,PTPRR^m, AJAP1^m, HS3ST2^m, and POU4F3^m for detecting CIN3⁺ and endometrial complex hyperplasia lesions was 100, 86, 71, 86, 86, 57, and 100%; specificity was 67, 79, 85, 50, 52, 96, and 52%, respectively. Testing for high risk-HPV had a sensitivity of 57% and specificity of 75% for CIN3+ and endometrial complex hyperplasia lesions.

Conclusion

Methylated (^m) SOX1^m and POU4F3^m could be new methylation biomarkers for detection of CIN3⁺ and endometrial complex hyperplasia in AGC. Women with AGC and positive SOX1^m / POU4F3^m, colposcopy, cervical conization or endometrial sampling should be considered.     

Helicobacter pylori infection can change the intensity of gastric Lewis antigen expressions differently between adults and children

This study tested whether there were different expressions of gastric Lewis antigens between children and adults with Helicobacter pylori infection, and whether the difference was related to the infection outcome. About 68 dyspeptic children and 110 dyspeptic adults were enrolled to check H. pylori infection, its colonization density, and the related histology. Gastric Lewis antigens b (Le^b), x (Le^x), and sialyl-Lewis x (sialyl-Le^x) were immunohistochemically stained and scored for the intensity. The H. pylori-infected adults, but not the children, had a lower Le^b intensity over the antrum (p = 0.019) but higher Le^b intensity over the corpus (p = 0.001) than the non-infected ones. Over the antrum, both the H. pylori-infected children and adults had a lower Le^x and higher sialyl-Le^x intensity than those non-infected ones (p < 0.05). The H. pylori-infected adults had a higher bacterial density (p = 0.004) and Le^b intensity (p = 0.016) over the corpus than the H. pylori-infected children. For the H. pylori-infected adults, but not children, the corpus had a higher Le^b (p = 0.038) and lower Le^x (p = 0.005) intensity than the antrum. Furthermore, the H. pylori-infected adults expressed a higher Le^b and had a higher bacterial density than those with weak Le^b (antrum, p < 0.001; corpus, p = 0.001). In conclusion, H. pylori infection is associated with the intensity change of Lewis antigen expressions in the stomach. The changes of gastric Lewis antigen expressions are different between adults and children with H. pylori infection, which may exert different H. pylori colonization over the corpus between adults and children.     

High-Density Expression of Ca2+-Permeable ASIC1a Channels in NG2 Glia of Rat Hippocampus

NG2 cells, a fourth type of glial cell in the mammalian CNS, undergo reactive changes in response to a wide variety of brain insults. Recent studies have demonstrated that neuronally expressed acid-sensing ion channels (ASICs) are implicated in various neurological disorders including brain ischemia and seizures. Acidosis is a common feature of acute neurological conditions. It is postulated that a drop in pH may be the link between the pathological process and activation of NG2 cells. Such postulate immediately prompts the following questions: Do NG2 cells express ASICs? If so, what are their functional properties and subunit composition? Here, using a combination of electrophysiology, Ca²⁺ imaging and immunocytochemistry, we present evidence to demonstrate that NG2 cells of the rat hippocampus express high density of Ca²⁺-permeable ASIC1a channels compared with several types of hippocampal neurons. First, nucleated patch recordings from NG2 cells revealed high density of proton-activated currents. The magnitude of proton-activated current was pH dependent, with a pH for half-maximal activation of 6.3. Second, the current-voltage relationship showed a reversal close to the equilibrium potential for Na⁺. Third, psalmotoxin 1, a blocker specific for the ASIC1a channel, largely inhibited proton-activated currents. Fourth, Ca²⁺ imaging showed that activation of proton-activated channels led to an increase of [Ca²⁺]_i. Finally, immunocytochemistry showed co-localization of ASIC1a and NG2 proteins in the hippocampus. Thus the acid chemosensor, the ASIC1a channel, may serve for inducing membrane depolarization and Ca²⁺ influx, thereby playing a crucial role in the NG2 cell response to injury following ischemia.     

Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ～30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ～0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.     

贵州30种杜鹃属植物叶背表皮显微形态特征

杜鹃属植物的分类历来都十分重视叶片表皮特征的分类学价值,无论是在各亚属之间还是在种及种下等级的划分中都很强调此类特征的作用。贵州省位于世界现代杜鹃分布中心边缘及向东部散布的过渡地带,省内自然分布的杜鹃种类资源丰富而又独具特色,包括很多尚未深入研究的特有类群。为进一步研究贵州杜鹃属植物的属下系统分类关系,该文对自然分布于贵州的30种杜鹃进行了叶背显微形态特征研究,其中有28种的叶背表皮特征为首次报道。研究材料全部来自采于贵州西北部百里杜鹃自然保护区内的杜鹃属植物,包括杜鹃亚属4种、糙叶杜鹃亚属1种、常绿杜鹃亚属22种、马银花亚属1种及映山红亚属2种,共计30种。研究方法为取成熟叶片处理后在JSM-6490型扫描电镜下对叶背表皮进行观测。叶背鳞片类型的划分方法参照前人工作。结果表明:这30种杜鹃叶片叶背表皮形态特征在电子显微镜下表现出明显的多样性,如有鳞类杜鹃具有鳞片而其他类群则无,叶表面有或无表皮毛,表皮毛排列稀疏或密生,气孔器未见或偶见,散生于乳突状或非乳突状突起之间,或角质层增厚气孔器下陷,内、外拱盖表面粗糙或光滑,气孔器周围有或无间断的条形突起,突起呈环状或羽状等,但在同一亚属或亚组中又呈现出或多或少的一致性。研究讨论表明,叶背表皮显微形态特征在杜鹃属植物的属下系统分类中具有重要的研究价值。     

A naphthoquinone and a lignan from the wood of Kigelia pinnata

A new naphthoquinone, kigelinone, and a new lignan, kigeliol, together with six known constituents including lapachol and dehydro-α- lapachone, were isolated from the wood of Kigelia pinnata. On the basis of spectral data and chemical degradations, kigelinone was characterized as 2-(1-hydroxyethyl)-8-hydroxy-naphtho[2,3-b]furano-4,9-dione and kigeliol as (2S,6S)-bis(3,4-methylenedioxyphenyl)- 3,7- dioxabicyclo[3.3.0]octan-1R,5R-diol or its enantiomer.