Molecular map of the Chlamydomonas reinhardtii nuclear genome

We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.     

Non-covalent thrombin inhibitors featuring P3-heterocycles with P1-bicyclic arginine surrogates

Novel, potent, and highly selective classes of thrombin inhibitors were identified, which resulted from judicious combination of P4-aromatics and P2-P3-heterocyclic dipeptide surrogates with weakly basic (calcd pKa approximately non-basic-8.6) bicyclic P1-arginine mimics. The design, synthesis, and biological activity of achiral, non-covalent, orally bioavailable inhibitors NC1-NC44 featuring P1-indazoles, benzimidazoles, indoles, benzotriazoles, and aminobenzisoxazoles is disclosed.     

Locally Synthesized Phosphatidylcholine, but Not Protein, Undergoes Rapid Retrograde Axonal Transport in the Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phosphatidylcholine in the sciatic nerve has been demonstrated only after injection of lipid precursors into the cell body region. We now report, however, that after microinjection (1 μl) of [methyl-³H]choline chloride into the rat sciatic nerve (35-40 mm distal to the L4 and L5 dorsal root ganglia), time-dependent accumulation of ³H-labeled material occurred in dorsal root ganglia ipsilateral, but not contralateral, to the injection site. The level of radioactivity in the ipsilateral dorsal root ganglia was minimal at 2 h after isotope injection but was significantly increased at 7, 24, 48, and 72 h after intraneural isotope injection (n = 3–8 per time point); at these time points, all of the radiolabel in the chloroform/methanol extract of the ipsilateral dorsal root ganglia was present in phosphatidylcholine. The radioactivity in the water-soluble fraction did not show a time-dependent accumulation in the ipsilateral dorsal root ganglia as compared with the contralateral DRGs, ruling out transport or diffusion of precursor molecules. In addition, colchicine injection into the sciatic nerve proximal to the isotope injection site prevented the accumulation of radiolabel in the ipsilateral dorsal root ganglia. Therefore, this time-dependent accumulation of radiolabeled phosphatidylcholine in the ipsilateral dorsal root ganglia is most likely due to retrograde axonal transport of locally synthesized phospholipid material. Moreover, 24 h after injection of both [³H]choline and [³⁵S]-methionine into the sciatic nerve, the ipsilateral/contralateral ratio of radiolabel was 11.7 for ³H but only 1.1 for ³⁵S. indicating that only locally synthesized choline phospholipids, but not protein, were retrogradely transported.     

Larvicidal activity of Acacia nilotica extracts and isolation of D-pinitol--a bioactive carbohydrate

A low-molecular-weight, sugar-like compound other than glucose, fructose, sucrose, or myo-inositol showing lipophilic nature was isolated from the EtOH extract of Acacia nilotica. The structure of the compound was determined by spectral methods. This alicyclic polyalcohol was found to be D-pinitol (= 3-O-methyl-D-chiro-inositol; 1). The configuration of the compound was confirmed by single-crystal X-ray analysis. The compound 1 is known from Soybean, Australian mangroves, Fagonia indica, Arachis hypogaea, etc., but we have isolated this compound for the first time from the aerial parts of A. nilotica. Very few references have been cited for compound 1 for its entomological activity, and there are no reports on mosquitoes. Therefore, the crude extracts of A. nilotica were tested for its biological activity against mosquito larvae. Acetone extract at 500-ppm concentration showed chronic toxicity against Aedes aegypti and Culex quinquefasciatus IVth instar mosquito larvae. Such a biological activity has been observed for the first time for this plant. This study could be a stepping stone to a solution for destroying larval species as well as consumption of such a widely grown, problem weed, A. nilotica. This larvicidal agent, since it is derived from plant, is eco-friendly, cost effective, non-hazardous to non-target organisms and would be safe unlike commercially available insecticides.     

Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size

The ecdysone receptor (EcR)-based gene regulation system is a tool for controlling gene expression. To improve the sensitivity of this system, we evaluated many two-hybrid format synthetic gene constructs in which the GAL4 DNA binding domain was fused to the ligand binding domain of the Choristoneura fumiferana EcR mutant V390I/Y410E (GEvy), and various activation domains--VP16, p53, p65, or E2F-i--were fused to the EF domains of chimeric human RXR. These gene switches were assayed in NIH3T3 cells, HEK293 cells, and in mouse quadriceps in the presence of the nonsteroidal inducer RG-115819 or GS-E. All of the two-hybrid format constructs had no or very low background in the "off" condition and high luciferase reporter gene expression levels in "on" conditions. Extremely high sensitivity was achieved, with EC50 values in the subnanomolar range and with maximal induction at 10 nM RG-115819. Co-expression of both receptor genes with encephalomyocarditis virus (EMCV) or eIF4G internal ribosome entry site (IRES) sequences gave robust induction levels. To reduce the size of the switch construct, we tested single receptor formats, in which any of 14 different activation domains were fused to GEvy. We identified several switches with acceptable levels of basal and maximal induction levels. The gene switches described here provide receptor configuration options suitable for gene function studies, therapeutic protein production in cell culture, transgenic mouse models, and gene/cell therapy.     

Gastric endocrine cells in rats with uremia and after thyroparathyroidectomy

The decrease in active kidney parenchyma amount causes disorders in hormone secretion processes and their inactivation failure. Experimental thyroparathyroidectomy is connected with an abrupt reduction in endocrine cells and hormones produced by them, which can be a stimulating factor as far as the increase and intensity of endocrine gastric cells activity is concerned. The aim of the study was the histomorphological and immunohistochemical evaluation of these cells in the gastric pylorus. Thyroparathyroidectomy was performed in rats 30 days after nephrectomy. Fragments of gastric pylorus were collected 14 days after the operation. Paraffin sections were stained with H+E and silver method. Immunohistochemical reactions were conducted using antibodies against calcitonin gene-related peptide (CGRP), somatostatin (ST), synaptophysin (SPh), neuron-specific enolase (NSE), and chromogranin (CgA). The results showed an increase in number of endocrine cells in stomachs of rats in experimental group as compared to controls. Endocrine cells were larger and contained more secretory granules.     

Synergistic enhancement of apoptosis by coralyne and paclitaxel in combination on MDA-MB-231 a triple-negative breast cancer cell line

Triple-negative breast cancer (TNBC) is the most outrageous subtype of breast cancer. Emphasizing the urge of new approach in cancer therapy, combinational drug therapy may be proven as an effective strategy. In our previous study, we reported that coralyne (COR) with paclitaxel (PTX) efficiently decreases the proliferation of MDA-MB-231 compared with MCF-7 cell line. Thus, we studied the effect of COR and PTX in combination on apoptosis of MDA-MB-231 cell line. In silico results demonstrated that COR intercalates DNA at a minor groove. In vitro approaches revealed that in combination (COR and PTX) increases the efficacy of apoptosis in MDA-MB-231 cell line by a significant increase in G1/S phase arrest, DNA fragmentation, and change in mitochondria membrane potential. The expression of ATM and ATR a serine/threonine-protein kinase, ataxia telangiectasia and Rad3-related protein were depleted with an increase in time from 24 to 48 hours in concurrent with increased levels of γH2AX indicating that DNA damage routes cells to enter apoptosis. This was confirmed by high levels of caspase-3 and cytochrome c. Also, the decrease in the expression levels of matrix metalloproteinase-9 confirmed the antimetastatic efficacy of COR + PTX. The present study indicates that the synergistic effect of COR and PTX can enhance apoptosis in MDA-MB-231 cell line and may be proven as a potential anticancer therapy against TNBC.     

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors

Background

Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.

Methods

In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSⁿ). The LC-ESI-MSⁿ of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.

Results

The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC).

Conclusion

Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles.