Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

Differentiation-Dependent Sialylation of Individual Neural Cell Adhesion Molecule Polypeptides During Postnatal Development

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.     

A simple haemolytic method for quantitation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from crude samples

A simple haemolytic assay method for quantitative estimation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from a crude preparation has been developed. The method has several advantages over mosquito-larvicidal methods of assay as it is inexpensive, highly sensitive and easier to run and can be used for performing a reasonably large number of assays rapidly with high precision and with a coefficient of variation that does not exceed 1.96%.     

Evidence for a correlation between ambient cholesterol levels and soluble plasma sialyltransferase enzyme activity

While soluble forms of the sialyltransferase (sialyl-T) enzyme have been detected in significant quantities in serum, the exact source(s) of the enzyme, or the factors controlling its secretion are poorly understood. In this study, we have examined the relationship between ambient plasma cholesterol concentrations and sialyl-T activities and also levels of constituent plasma sialoglycoproteins (SGP). There was an inverse relationship between levels of the 2,6 sialyl-T enzyme and both total plasma cholesterol and HDL, although no such relationship was observed for the 2,3 enzyme. While there was no correlation between total cholesterol and the levels of plasma SGPs, there was an inverse relationship between the HDL component and 2,3 SGPs.     

On the defence strategy of Physa fontinalis (L.), a freshwater pulmonate snail

Summary Physa fontinalis (L.) gives a characteristic, chemically mediated escape response when stimulated by the majority of British leeches and flatworms. The snail responds rapidly and consistently to contact with all the molluskivorous leeches but also to three species which may be considered harmless. However, no response was given to Erpobdella octoculata, the most abundant and widespread of the harmless leeches. The flatworms generally evoked less strong reactions. The adaptive significance of the pattern of responsiveness is discussed. A weaker shell-shaking response is elicited in conspecifics and it is shown that this antisocial behaviour leads to a relatively spaced-out dispersion pattern. A possible adaptive advantage is the reduction of risk of detection by shell-crushing fish predators, to which the snails are otherwise extremely vulnerable.     

The influence of cell elastic modulus on inertial positions in Poiseuille microflows

Microchannels are used as a transportation highway for suspended cells both in vivo and ex vivo. Lymphatic and cardiovascular systems transfer suspended cells through microchannels within the body, and microfluidic techniques such as lab-on-a-chip devices, flow cytometry, and CAR T-cell therapy utilize microchannels of similar sizes to analyze or separate suspended cells ex vivo. Understanding the forces that cells are subject to while traveling through these channels are important because certain applications exploit these cell properties for cell separation. This study investigated the influence that cytoskeletal impairment has on the inertial positions of circulating cells in laminar pipe flow. Two representative cancer cell lines were treated using cytochalasin D, and their inertial positions were investigated using particle streak imaging and compared between benign and metastatic cell lines. This resulted in a shift in inertial positions between benign and metastatic as well as treated and untreated cells. To determine and quantify the physical changes in the cells that resulted in this migration, staining and nanoindentation techniques were then used to determine the cells’ size, circularity, and elastic modulus. It was found that the cells’ exposure to cytochalasin D resulted in decreased elastic moduli of cells, with benign and metastatic cells showing decreases of 135 ± 91 and 130 ± 60 Pa, respectively, with no change in either size or shape. This caused benign, stiffer cancer cells to be more evenly distributed across the channel width than metastatic, deformable cancer cells; additionally, a decrease in the elastic moduli of both cell lines resulted in increased migration toward the channel center. These results indicate that the elastic modulus may play more of a part in the inertial migration of such cells than previously thought.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.     

Loss of small glaciers will diminish beta diversity in Pyrenean streams at two levels of biological organization

Aim Small (< 1 km²) alpine glaciers are likely to disappear in this century, resulting in decreased regional habitat heterogeneity in associated streams. Both heterogeneity within and spatial isolation among glacier‐influenced streams can enhance beta diversity of stream‐dwelling organisms. We measured beta at both community and population‐genetic levels within and among streams currently influenced by small Pyrenean glaciers. We aimed to evaluate whether patterns are analogous between the two levels, to apply various approaches for characterizing beta, and to infer the outcome of future glacier loss on regional biodiversity. Location Four glacier‐fed basins in the Parc National des Pyrénées, France. Methods We classified each of 18 stream reaches across the basins into either high‐, mid‐ or low‐‘glaciality’ (glacial influence) groups according to four physicochemical characteristics. At each reach, we collected macroinvertebrate communities and evaluated mitochondrial DNA haplotypes for 11–13 individuals of Baetis alpinus Pictet. Using taxa/haplotypes as basic units, we evaluated community and population‐genetic beta diversity simultaneously. We measured beta diversity in three major ways: as multivariate (Sørensen's dissimilarity, Jost D) and ‘classical’ (gamma/alpha) variation to compare among glaciality groups, and as turnover along the glaciality gradient within each basin. Results For most approaches at both organizational levels, beta was greatest among high‐glaciality reaches, absolute values of variation of beta in high‐glaciality streams were strikingly similar between levels, and the steepest turnover within basins occurred between high‐ and mid‐glaciality reaches. Therefore, high‐glaciality reaches contained assemblages and populations that were unique both within that stream type (among basins) and compared with other stream types within basins. Main conclusions Parallel beta diversity patterns at population‐genetic and community levels suggested that environmental drivers influence these levels analogously. Extreme conditions (e.g. low temperature, high instability, isolation) in high‐glaciality streams probably enhance beta at both levels. Stream beta diversity is likely to decrease substantially with continued glacial reduction in this system.