Silencing of FABP3 Inhibits Proliferation and Promotes Apoptosis in Embryonic Carcinoma Cells

Fatty acid–binding protein 3 (FABP3) facilitates the movement of fatty acids in cardiac muscle. Previously, we reported that FABP3 is highly upregulated in the myocardium of ventricular septal defect patients and overexpression of FABP3 inhibited proliferation and promoted apoptosis in embryonic carcinoma cells (P19 cells). In this study, we aimed to investigate the effect of FABP3 gene silencing on P19 cell differentiation, proliferation and apoptosis. We used RNA interference and a lentiviral-based vector system to create a stable FABP3-silenced P19 cell line; knockdown of FABP3 was confirmed by quantitative real-time PCR. Expression analysis of specific differentiation marker genes using quantitative real-time PCR and observation of morphological changes using an inverted microscope revealed that knockdown of FABP3 did not significantly affect the differentiation of P19 cells into cardiomyocytes. CCK-8 proliferation assays and cell cycle analysis demonstrated that FABP3 gene silencing significantly inhibited P19 cell proliferation. Furthermore, Annexin V-FITC/propidium iodide staining and the caspase-3 activity assay revealed that FABP3 gene silencing significantly promoted serum starvation–induced apoptosis in P19 cells. In agreement with our previous research, these results demonstrate that FABP3 may play an important role during embryonic heart development, and that either overexpression or silencing of FABP3 will lead to an imbalance between proliferation and apoptosis, which may result in embryonic cardiac malformations.     

Ring A rearranged limonoids from the fruits of Aphanamixis grandifolia and their cytotoxicity evaluation

Two new limonoids aphanamolides C (1) and D (2), together with two known limonoids aphanamolide A (3) and aphapolynin A (4), were isolated from the fruits of Aphanamixis grandifolia. Their structures were assigned on the basis of spectroscopic data, with the absolute configurations of 1 and 2 being established by electronic circular dichroism (ECD) spectroscopic analyses. Those limonoids varied in the ring A: aphanamolide C featured two oxygenated bridges, and aphanamolide D was the second example containing β-hydroxy-α,β:γ,δ-dienoate moiety. The cytotoxic activities were also evaluated in vitro against four human cancer cell lines (MCF-7, A549, SMMC-7721, and HL-60).     

Molecular Mechanisms Underlying the Analgesic Property of Intrathecal Dexmedetomidine and Its Neurotoxicity Evaluation: An In Vivo and In Vitro Experimental Study

Background

Dexmedetomidine (DEX) has been used under perioperative settings as an adjuvant to enhance the analgesic property of local anesthetics by some anesthesiologists. However, the analgesic mechanisms and neurotoxicity of DEX were poorly understood. This study examined the effect of DEX alone on inflammatory pain, and it also examined the underlying molecular mechanisms of DEX in the spinal cord. Furthermore, in vivo and in vitro experiments were performed to investigate the neurotoxicity of DEX on the spinal cord and cortical neurons.

Methods

This study used adult, male Kunming mice. In the acute inflammatory model, the left hind-paws of mice were intradermally injected with pH 5.0 PBS while chronic constrictive injury (CCI) of the sciatic nerve was used to duplicate the neuropathic pain condition. Thermal paw withdrawal latency and mechanical paw withdrawal threshold were tested with a radiant heat test and the Von Frey method, respectively. Locomotor activity and motor coordination were evaluated using the inverted mesh test. Western blotting examined spinal ERK1/2, p-ERK1/2, caspase-3 and β-actin expressions, while spinal c-Fos protein expression was realized with immunohistochemical staining. Hematoxylin eosin (HE) staining was used to examine the pathological impacts of intrathecal DEX on the spinal cord. DAPI (4′,6-diamidino-2-phenylindole) staining was used to observe cell death under an immunofluorescence microscope.

Results

Intra-plantar pH 5.0 PBS-induced acute pain required spinal ERK1/2 activation. Inhibition of spinal ERK1/2 signaling by intrathecal injection of DEX displayed a robust analgesia, via a α2-receptor dependent manner. The analgesic properties of DEX were validated in CCI mice. In vivo studies showed that intrathecal DEX has no significant pathological impacts on the spinal cord, and in vitro experiments indicated that DEX has potential protective effects of lidocaine-induced neural cell death.

Conclusion

Intrathecal injection of DEX alone or as an adjuvant might be potential for pain relief.     

Cyanobactericidal Effect of Streptomyces sp. HJC-D1 on Microcystis auruginosa

An isolated strain Streptomyces sp. HJC-D1 was applied to inhibit the growth of cyanobacterium Microcystis aeruginosa FACHB-905. The effect of Streptomyces sp. HJC-D1 culture broth on the cell integrity and physiological characteristics of M. aeruginosa FACHB-905 was investigated using the flow cytometry (FCM), enzyme activity and transmission electron microscopy (TEM) methods. Results showed that the growth of M. aeruginosa FACHB-905 was significantly inhibited, and the percentage of live cells depended on the culture broth concentration and exposure time. The activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) increased with exposure concentration and exposure time, and the significant increase of reactive oxygen species (ROS) led to the disruption of the subcellular structure of M. aeruginosa FACHB-905, and caused the increase of malondialdehyde (MDA). Furthermore, TEM observation suggested the presence of three stages (cell breakage, organelle release and cell death) for the cyanobactericidal process of Streptomyces sp. HJC-D1. Therefore, Streptomyces sp. HJC-D1 not only affected antioxidant enzyme activities and ROS level, but also destroyed the subcellular structure of M. aeruginosa FACHB-905, demonstrating excellent cyanobactericidal properties.     

Complete Genome Sequence of the First Chinese Virulent Infectious Laryngotracheitis Virus

Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (U_L) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs.     

Development of Simple Sequence Repeat Markers from De Novo Assembled Transcriptomes of Pumpkins

Plant Molecular Biology Reporter - Pumpkin (Cucurbita spp.) is one of the major vegetable crops grown worldwide. The number of simple sequence repeat (SSR) markers in pumpkins lags far behind the...     

Pseudomembranous Candidiasis in HIV/AIDS Patients in Cali, Colombia

Candida albicans is the most frequently isolated yeast from the oral cavity of HIV/AIDS individuals. The use of fluconazole has increased the number of resistant or less-sensitive Candida species different than C. albicans. The purpose of this study was to identify the Candida species producing pseudomembranous candidiasis in patients suffering from AIDS, their relationship with CD4⁺ counts and their sensitivity to fluconazole and itraconazole. We studied 71 patients at a hospital in the city of Cali. Samples of white plaque were seeded on CHROMagar Candida, yeast identification was done with API 20C Aux, and susceptibility testing was determined by E test. Ninety-three yeast isolates were obtained, 52 single and 41 mixed. C. albicans was the most isolated, followed by C. glabrata. An increased frequency of isolates and variety of Candida species occurred in patients with a CD4⁺ cell count ≤100 cells/mm³ without significant differences (p = 0.29). The susceptibility study showed that 8 (8.6 %) isolates were resistant to fluconazole and 11 (11.8 %) to itraconazole, while 6 (8.8 %) C. albicans were simultaneously resistant. No association was found between the isolates of C. albicans or Candida species different than C. albicans and the use of fluconazole (p = 0.21). The results of this study indicate that in the tested population, fluconazole continues to be the best treatment option for oropharyngeal candidiasis in patients suffering from AIDS (HIV/AIDS); however, susceptibility tests are necessary in patients who present therapeutic failure.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.