Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Export of Galectin-3 from Nuclei of Digitonin-Permeabilized Mouse 3T3 Fibroblasts

Galectin-3 is a galactose-/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. Immunofluorescence staining revealed that galectin-3 distributes differentially between the nucleus and the cytoplasm, depending on the proliferative state of the cells under analysis. Using digitonin-permeabilized mouse 3T3 fibroblasts, we provide evidence that galectin-3 is rapidly and selectively exported from the nucleus. Although both phosphorylated and nonphosphorylated isoforms of galectin-3 are found in the nuclear fraction, only phosphorylated galectin-3 is identified in the exported fraction, implying that phosphorylation is important for the nuclear export of the protein. The rate of galectin-3 export is temperature dependent and is decreased by the addition of wheat germ agglutinin. More strikingly, galectin-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1). Indeed, a putative leucine-rich nuclear export signal can be found in residues 241-249 of the murine galectin-3 sequence. Finally, gel filtration of the exported material showed that galectin-3 can be found in at least two high molecular weight complexes (approximately 650 and approximately 60 kDa), both of which can be disrupted by lactose.     

Isolation and characterization of phytochelatin synthase in rice seedlings

Rice plants were treated with 50 microM copper sulfate to induce the synthesis of phytochelatins by means of a series of enzymatic reactions, including that of photochelatin synthase. Phytochelatin synthase extracted from 3-week-old rice seedlings was purified through a series of steps including precipitation with ice-chilled acetone, QAE A-50 anion exchange column, Amicon XM-50 ultrafiltration and Polybuffer Exchange (PBE) 94 chromato-focusing. This enzyme had a molecular mass of about 100 kDa with an isoelectric point of 4.0. The temperature and pH optima of this enzyme were 55 degrees C and pH 7.5, respectively. The enzyme was thermal tolerable and unstable under refrigeration at 4 or -20 degrees C. Cadmium was the most effective stimulator, followed by lead, copper, silver, cobalt and other divalent cations. Calcium and magnesium had no effect.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

HPTN 062: A Pilot Randomized Controlled Trial Exploring the Effect of a Motivational-Interviewing Intervention on Sexual Behavior among Individuals with Acute HIV Infection in Lilongwe,Malawi

Objective

We pilot tested a Motivational Interviewing (MI) –based counseling intervention for individuals with Acute HIV Infection (AHI) to reduce risky sexual behavior in Lilongwe, Malawi.

Methods

Twenty-eight individuals diagnosed with AHI were randomized to receive either brief education alone, or the brief education plus the MI-based intervention, called Uphungu Wanga. Participants in Uphungu Wanga received four sessions delivered on the day of diagnosis, three days later and at weeks 1 and 2 with a booster session at week 8; participants were followed for 24 weeks from diagnosis. An interviewer administered quantitative questionnaire was conducted at baseline and at weeks 2, 4, 8, 12, 16, 20 and 24. Semi-structured qualitative interviews (SSI) were conducted at weeks 2, 8, 12, and 24.

Results

The majority of participants in both arms reported rapid and sustained behavior change following diagnosis with AHI. Very few participants reported having sex without a condom after diagnosis. Participants reported a trend towards fewer sex partners and abstaining from sex during study follow-up. Participants in the MI-based arm provided concrete examples of risk reduction strategies in the SSIs while those in the brief education arm primarily described reducing risk behavior, suggesting that the MI-based group may have acquired more risk reduction skills.

Conclusions

Individuals in both study arms reduced risky sexual behaviors after diagnosis with AHI. We found few major differences between study arms during the 6-month follow up period in self-reported sexual behaviors therefore a MI-based intervention may not be needed to trigger behavior change following AHI. However, comparing the MI-based intervention to repeated brief education sessions made it difficult to assess the potential benefit of an MI-based intervention in a setting where standard counseling often consists of one post-test session. Nevertheless, provision of counseling immediately following diagnosis with HIV to support behavior change should remain a priority.

Trial Registration

ClinicalTrials.gov NCT01197027     

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Background

Parkinson’s disease is the second most common neurodegenerative disorders after Alzheimer’s disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration.

Results

Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP⁺) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP⁺ impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP⁺ might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP⁺ caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP⁺ decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP⁺ provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP⁺.

Conclusions

Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson’s disease.