Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester

Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.     

Microfluorometric Evaluation of Calcein Acetoxymethyl Ester as a Probe for P-Glycoprotein-Mediated Resistance: Effects of Cyclosporin A and Its Nonimmunosuppressive Analogue SDZ PSC 833

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.     

Tissue Culture of Lily Filament and Its Cytological Observations

Anthers with the filament of lily （Lilium davidii var. Willmottiae (Wilson) Roffill） were planted on modified MS medium. Filament cultures were incubated at 25℃± 1℃, and illuminated with a fluorescent light of about 800–1200 lux. Cultured filaments were initially short in length, but gradually became thicker and elongater after 20 days. After 30 days, the calli (about 2 mm in size) were produced. The calli were then transferred to the differentiation medium. After 20 days, the bulblets were developed from the calli, and the regeneration plants were formed after 40 days for tetraploid induction, plantlets were treated by aqueous solution of colchicine. A preliminary ploidy analysis of the root tip cells of these treated plants indicated that it was predominantly tetraploid (4n=48) Of 56 mitotic figures examined, 19 were tetraploid, 10 were diploid and 27 were aneuploid. The intercellular migrating chromatin substance appeared in the calli were also observed.     

Cytological Observations of Microsporogenesis and Pollen Development in Wheat in Vivo

Cytological observations of microsporogenesis and pollen development in vivo in wheat were carried on by means of phase contrast optics, which could avoid the distortions resulted from materials difficult to be fixed. The cytological changes were observed as follows: 1. Just before first mitosis of pollen, many strands of cytoplasm arose from one side of the nucleus facing the aperture, and moved swingingly toward the aperture. And then these strands of cytoplasm combined into one mass and protruded in the large vacuole. 2. There was a alteration in the direction of the spindle axis from obliquity to parallelism in anaphase. 3. Forming course of the wall between generative and vegetative cell. 4. Dynamic course of the disintegration of wall between vegetative and generative cell as well as that the generative cell came into the vetetafive cell.     

Cytological Observations on Some Species of Dryopteris and Polystichum from China

Cytological observations on five Dryopteris species and four Polystichum species from China are reported. Most of the materials examined were field-fixed in Emei Shan, Sichuan province, and one of them was in Dali Xian, Yunnan province. Only the count of Dryopteris goeringiana was made from the plant brought from Wuling Shan, Beijing and grown in the garden of our Institute. All the materials were fixed in 1:3 acitic acidalcohol, and stained with acetocamine. All the counts were made at diakinesis or metaphase I of meiosis in SMCs .The results of the observation on them are summarized in table 1. Of these, the chromosome numbers for six species are first report. Five species are sexual: diploid: D. goeringiana, D. porosa, D. subinaequalis, D. yui and P. stenoghyllum. Three spe cies are sexual tetraploid: P. acutidens, P. erosum and P. moupingense. Only D. juxtaposita is an apomictic triploid. The vouchers are preserved in the Herbarium of our Institute (PE). We are indebted to Prof. R. C. Ching and C. R. Fraser-Jenkins for identification of the materials.     

Cytological Observations on Microsporogenesis and Pollen Development of Regenerated Stamen in Hyacinth

The present study consists of the cytological observations on the process of microsporogenesis and pollen development in the regenerated stamen of hyacinth; and a comparative study of the cytological changes in stamens of both regenerated and produced under natural condition. Results showed that the cytological changes of microsporogenesis and the pollen deveLopment in the regenerated stamen of the hyacinth were basically normal. But in the stage of the mature pollen there was an obvious cytological difference between both stamens in vitro and in nature. The mature pollen of the regenerated stamen consisted of three cells: one vegetative cell and two sperms, while mature pollen grain under natural condition was made up of two cells: one vegetative cell and one generative cell. This difference mainly resulted from different time and place of the generative cell division. The reason resulting in the differences and their influence on sperms were discussed.     

Cytological Observations on Three British Species of Laminaria : a Preliminary Report

Mitotic divisions in male and female gametophytes and in youngsporelings of Laminaria digitata, L. ochroleuca, and L. saccharinafrom Plymouth follow a normal course and do not show the ‘endomitoticmultiplication’ of chromosomes described by Walker inL. digitata from Scotland. Chromosome counts on all three speciesalmost invariably give a value for n between 27 and 31, anda definite count of this order will probably emerge.     

The Test Structure and Composition of the Foraminifer Rosalina floridana*

SYNOPSIS. The composition of the test of Rosalina floridana (Cushman) was examined histochemically, and its structure was studied with the electron microscope by means of thin sections and carbon replicas. The test is composed of a thick organic lining overlain by one or more calcite layers bounded above and below by thin membranes. The membranes are fused to organic pore processes composed of coarse fibers that penetrate the calcite layers. The ***lining, consisting of coarse fibers matted into a laminated sheet, is considered a strengthening element of the test. The membranes covering each calcite layer are composed of fine, headed fibrils which in aggregate have a striated pattern; they are thought to be the crystal-nucleating agent during calcification and to form a protective covering for the previously deposited calcite layers. The pore processes, which are devoid of an internal entrance for cytoplasm, are considered to be points of attachment for the membranes; they tie the organic test components into a unified whole. The calcite layers and the chambers lack this unity, being separated from each other and from the preceding chambers by membranes so that there are no calcite-to-calcite boundaries between them. An organic, sievelike structure of undetermined function has been found in the foramina of chambers near the prolocular region of the test. Histochemical methods show that the lining contains proteins, polysaccharides, and unidentified substances; the membranes and the pore processes stain as a protein-polysaccharide complex free of other substances.     

Use of Dissolved Amino Acids by the Foraminifer Notodendrodes antarctikos

SYNOPSIS. Foraminifera are a ubiquitous and sometimes numericallyimportant component of benthic communities. This paper discussesthe role of free amino acids in the nutrition of Notodendrodesantarctikos, a large arborescent foraminifer from an oligotrophicembayment of the Ross Sea, Antarctica. The effects of temperatureand substrate concentrations suggest a carrier mediated transportsystem which facilitates the accumulation of a wide varietyof free amino acids at concentrations found in the interstitialwaters of its sedimentary habitat. Involvement of isotopicallylabeled amino acids in the metabolism of this organism is discussed.     

试析中国长江中游地区大型尖状类石器遗存的区域传统

大型尖状类石器在长江中游地区相邻或相近的汉水上游、丹江口库区、三峡地区和澧阳平原等不同区域保存有不同的技术传统。汉中盆地的大型尖状类石器多选自河滩砾石为毛坯直接加工。丹江口库区无论是调查数据还是发掘数据其比例均高于汉中盆地,现有发掘材料来看其大型尖状类石器有稳定的传统,即选自砾石直接单面或两面加工而成,但调查的材料显示存在另外一种传统,即选取砾石生产长度大于10cm的大石片,以大石片为毛坯加工大型尖状类石器。三峡地区古人类在更新世期间似乎不存在有生产大型尖状类石器的传统,为数不多的手镐与当地的砾石砍砸器和石片砍砸器应当为"同质异型"类器物。澧阳平原及周边丘陵区内发现的大型尖状类石器相比于汉中盆地和丹江口库区,发现数量最多,技术传统也最为复杂,至少包括三种不同的技术。大型尖状类石器在不同区域显示不同的技术传统,暗示从中更新世开始长江中游地区可能在一定程度上形成了并不简单的文化关系。     

Cytological Observations on Nodules,Nodule-Like Structures and Crown Galls on the Roots of Phaseolus vulgaris

The transconjugant of Agrobacterium tumefaciens with C58 genetic background containing plasmid pSym3622 of Rhizobium leguminosarum by. phaseoli was capable of infecting the roots of Phaseolus vulgaris of the wounding sites and inducing the formation of nodule-like structures near the sites of inoculation. These structures were different from the nodule and crown galls induced respectively by R. leguminosarumbv, phaseoli 3622--15 and a virulant strain A208 (pTiT37) of A. tumefaciens. The vascular bundles of the nodule-like structures were located at the central region of the structures and were surrounded by highly vacuolated thin-walled cells rich in starch grains. There was no bacterium neither in cell nor in the intercellular space of these structures evidenced by light and electron microscopic observations. In contrast, many metabolically active bacteroids were observed within the cells and in the intercellular spaces of the effective nodules, in the case of crown galls, there was also no bacterium in any part of the tumours. The internal structures of tumour on the root of Phaseolus vuigaris were visualized as distinguishable coherent areas of cells partitioned by disorganized cell zones. These areas of cells were traced back to be derived from different meristematic zones. At some instance, a number of root-like protrusions which were likely to develop into new roots were observed. In other parts, host cells were visible as a result of cell degeneration.     

蕨受精作用的细胞学观察

为了阐明进化蕨类受精作用的特点和细胞学机制,该文采用透射电镜观察了蕨(Pteridium aquilinum var.latiusculum)受精作用的主要过程,观察结果显示:(1)蕨精子通过受精孔进入卵细胞,多数情况下,该精子的螺旋运动先在受精孔的下方产生一个受精腔,然后精子再与卵细胞质融合。(2)第一个精子的这种延迟的螺旋运动和因精子的钻入而引起的卵细胞固缩反应可能是阻止多精受精的重要因素。(3)卵发育时期产生的核外突在受精后仍能持续12 h,然后与核本体分离,逐渐在细胞质中消解。(4)合子通过其后方细胞质的液泡化而建立了水平极性,此后再进行细胞分裂。该研究观察到了进化蕨类受精作用过程中的一些新现象,包括产生受精腔、卵细胞固缩反应、核外突的命运以及合子极性建立等,这有助于理解蕨类植物的受精作用机制及有性生殖的演化。     

Biotinylation of Oligonucleotides Using a Water Soluble Biotin Ester

Abstract

Oligonucleotides aminated at the 5′-end were bio-tinylated with a water soluble N-hydroxysuccinimide ester of biotin in large scale. The biotinylated oligonucleotides were purified by reversed phase HPLC.     

The Process of Chamber Formation in the Foraminifer Rosalina floridana (Cushman)

SYNOPSIS. The foraminifer Rosalina floridana builds a chambered, calcareous test which is periodically enlarged by the addition of new chambers. R. floridana begins to form a chamber by constructing an algal growth cyst which covers the dorsal side of the animal and is cemented to the substrate. The pseudopods that build the cyst coalesce to form a cytoplasmic template or anlage on which the chamber walls will be secreted. Electron micrographs reveal the anlage cytoplasm to be a “froth” of nearly empty vesicles which contain mitochondria, fibrillar material, and electron dense granules. The organic lining of the new chamber is secreted on the anlage by pseudopods extending thru it. After the organic lining is completed, cytoplasm from within the test flows into the forming chamber and forces the anlage cytoplasm out thru the new aperture. This “frothy” anlage cytoplasm forms a sheath over the dorsal surface of the test; while it is in place, a layer of calcite is deposited on the walls of the new chamber and over the rest of the test. When calcification is completed, the sheath breaks up and is incorporated into newly formed pseudopods as the foraminifer gradually moves out of the growth cyst and begins normal feeding. The production of vesiculated cytoplasm in normal pseudopods and in the anlage is viewed as a method of greatly increasing cytoplasmic volume with a resultant very small loss in cytoplasmic mass. In the anlage during the production of the organic lining the vesiculated cytoplasm apparently acts only as a support for the membranes being secreted by the pseudopods and presumably does not take part in the secretory process. This same cytoplasm forms the sheath that is present during calcification. The view is advanced that, altho the sheath could be active during calcification with the mitochondria within the vesicles actively transporting calcium to sites of crystal growth, its more probable function is to form a partition between the parts to be calcified and the environment. The foraminifer could then secrete CaCO₃ from pooled reserves in the cytoplasm into the area between the sheath and the chamber membranes. The basal membrane is considered the nucleating agent during calcification and is responsible for the ordering of the crystals so their C axes are perpendicular to the surface of the chamber.     

Cytological Observations of the Infection Process by Phomopsis helianthi (Munt.-Cvet) in Leaves of Sunflower

The infection process of Phomopsis helianthi and the specific degradation of infected tissue were studied in detail using light and transmission electron microscopy. In comparison with other vascular pathogens, the infection and degradation process was in some aspects different. The favourite tissue for the pathogen to grow in was the phloem. Parenchymatic cells in and around vascular bundles were extremely sensitive to infection long before hyphae arrived, probably due to a toxin. In the parenchymatic cells the first changes were visible at the chloroplasts where electron-dense material accumulated in the thylakoid space. The chloroplast stroma changed contrast and later the whole cytoplasm also appeared electron dense. In the vascular bundles, first the phloem was destroyed and then hyphae invaded the adjacent mesophyll, the cambium, and finally the vessel elements. In particular, the compact mesophyll of the midvein was severely affected. Vessel elements were lined with electron-dense material and some were filled with flocculent material. Severe wall destruction indicated the action of a complete set of cell wall-degrading enzymes before hyphae entered the tissue; it always started at the innermost wall layer. Wall degradation in vascular tissue and adjacent parenchyma with intercellular spaces was different. Before the degradation of the protoplasts started, the cell walls were completely metabolized and only the secondary walls of the vessels resisted for longer. There were no host–cell reactions visible that could be interpreted as a defence reaction.     

Evolution of a Planktonic Foraminifer during Environmental Changes in the Tropical Oceans

Ecological adaptation to environmental changes is a strong driver of evolution, enabling speciation of pelagic plankton in the open ocean without the presence of effective physical barriers to gene flow. The tropical ocean environment, which plays an important role in shaping marine biodiversity, has drastically and frequently changed since the Pliocene. Nevertheless, the evolutionary history of tropical pelagic plankton has been poorly understood, as phylogeographic investigations are still in the developing state and paleontological approaches are insufficient to obtain a sequential record from the deep-sea sediments. The planktonic foraminifer Pulleniatina obliquiloculata is widely distributed in the tropical area throughout the world’s oceans, and its phylogeography is well established. It is thus one of the best candidates to examine how past environmental changes may have shifted the spatial distribution and affected the diversification of tropical pelagic plankton. Such an examination requires the divergence history of the planktonic foraminifers, yet the gene marker (partial small subunit (SSU) rDNA) previously used for phylogeographic studies was not powerful enough to achieve a high accuracy in estimating the divergence times. The present study focuses on improving the precision of divergence time estimates for the splits between sibling species (genetic types) of planktonic foraminifers by increasing the number of genes as well as the number of nucleotide bases used for molecular clock estimates. We have amplified the entire coding regions of two ribosomal RNA genes (SSU rDNA and large subunit (LSU) rDNA) of three genetic types of P. obliquiloculata and two closely related species for the first time and applied them to the Bayesian relaxed clock method. The comparison of the credible intervals of the four datasets consisting either of sequences of the partial SSU rDNA, the complete SSU rDNA, LSU rDNA, or a combination of both genes (SSU+LSU) clearly demonstrated that the two-gene dataset improved the accuracy of divergence time estimates. The P. obliquiloculata lineage diverged twice, first at the end of the Pliocene (3.1 Ma) and again in the middle Pleistocene (1.4 Ma). Both timings coincided with the environmental changes, which indirectly involved geographic separation of populations. The habitat of P. obliquiloculata was expanded toward the higher latitudinal zones during the stable warm periods and subsequently placed on the steep environmental gradients following the global cooling. Different environmental conditions in the stable warm tropics and unstable higher latitudes may have triggered ecological divergence among the populations, leading to adaptive differentiation and eventually speciation. A comprehensive analysis of divergence time estimates combined with phylogeography enabled us to reveal the evolutionary history of the pelagic plankton and to find the potential paleoenvironmental events, which could have changed their biogeography and ecology.     

The Cytological Diagnosis of Nasopharyngeal Carcinoma Using A Silk Swab Stick

A prospective study was carried out to investigate the efficacy of exfoliative cytology for the diagnosis of nasopharyngeal carcinoma. Exfoliated nasopharyngeal cells were collected from patients with a clinical suspicion of carcinoma by scraping the nasopharyngeal mucosa with a piece of silk wrapped around one end of a wooden stick. Ninety-eight patients were investigated in this manner, and the results of cytological investigation compared with the histological findings. The sensitivity of cytological investigation was 71% and the overall accuracy was 86%. The combined use of cytology and serology as a method of screening for nasopharyngeal carcinomas is discussed.     

Enzymatic Oligomerization of the Tetrapeptide Ester Allylglycine-Phenylalanine-Phenylalanine-Allylglycine Ethyl Ester

The native hydrolytic action of subtilisin Carlsberg was reversed to oligomerize the tetrapeptide ester L-Ag-L-Phe-L-Phe-L-Ag-OEt, containing the unnatural amino acid allyglycine (Ag, where R = -CH₂-CH = CH₂), in several miscible aqueous/organic solvent systems. Mass spectrometry indicated that the octapeptide ester (Ag-Phe-Phe-Ag)² OEt was formed in all cases and that the dodecapeptide ester (Ag-Phe-Phe-Ag)₃OEt was formed in one case. Additional mass spectrometry peaks indicated that a number of other peptides larger than the octapeptide ester (Ag-Phe-Phe-A)²OEt may also have been formed. Infrared analysis revealed that the oligopeptide products exhibit P-sheet peptide folding.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.