Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.     

Expression of motility in strains of the non-motile species Aeromonas media

Abstract Four isolates of the non-motile species Aeromonas media , icluding the type strain, were demonstrated by transmission electronk microcopy to possess polar flagella. Motility appeared to be restricted to old, regularly subcultured strains, and coincided with an increase in colony size and a concomitant reduction in the amount of brown diffusible pigment produced by colonies on tryptone soya agar.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Diethylpyrocarbonate—An effective agent for the sterilization of different types of nutrient media

A simple and convenient method has been tested for the steriltzation of nutrient media for long-term cultivation of plant cells. Diethylpyrocarbonate is suitable for this task in concentrations about 1000 mg l^-1 The cells cultivated for 15 subsequent passages on media treated by DPC had the same growth parameters, production pattern and ability to transform exogeneous organic compounds as did the controls. The method is suitable for the preparatian of both liquid and agar media, for stabilization of stock solutions and for sterilization of cultivation vessels and tubing.Abbreviations DPC diethylpyrocarbonate - medium MS nutrient medium according to Murashige and Skoog - NAA naphtaleneacetic acid     

Oxygen transfer deficiencies in starch-based media

Insufficient oxygen transfer in a highly viscous media with varying viscosity occurs during the degradation of starch byBacillus licheniformis. Oxygen transfer rate (OTR) decreased below 0.3 gl^–1 h^–1 for viscosities above 5 to 10 mPa.s and agitation speeds lower than 710 rev/min. Increasing agitation speeds by 30 to 50% compensated for the decrease in OTR for viscosities between 10 and 200 mPa.s. A dual impeller is considered essential for growth improvement.     

紫晶香蘑栽培生物学研究

紫晶香蘑栽培生物学研究卢成英，李鹄鸣，钟以举（湖南省吉首大学生态研究所４１６０００）ＢｉｏｌｏｇｉｃａｌＲｅｓｅａｒｃｈｏｎＬｅｐｉｓｔａｓｏｒｄｉｄａＣｕｌｔｉｖａｔｉｏｎ．￥Ｌｕｃｈｅｎｇｙｉｎｇ；ＬｉＨｕｍｉｎｇ；ＺｈｏｎｇＹｉｊｕ（Ｉｎ－ｓｔ...     

Different sensitivities of the sugar receptor of the lateral styloconic sensillum in fourth- and sixth-instar larvae of the spruce budworm Choristoneura fumiferana

Female fourth- and sixth-instar larvae, Choristoneura fumiferana, were tested individually for the response of the sugar cell on the lateral styloconic sensillum to 25 mM/l concentrations of 12 carbohydrates. The spruce budworm showed an age-related change in responsiveness of the sugar cell. The order of stimulating effectiveness for fourth-instars was melibiose > sucrose > raffinose.These storage di- and trisaccharides are present in the host plant at the beginning of budbreak. Sixth-instars responded to sucrose > fructose> m-inositol. These findings are in accordance with those of a previous behavioural study on feeding preferences of sixth-instars. The response for both melibiose and raffinose does not change from fourth- to sixth-instars; however, it does for sucrose, fructose and m- inositol.