Arabidopsis antiporter CHX23 and auxin transporter PIN8 coordinately regulate pollen growth.

Both the antiporter CHX23 (Cation/Proton Exchangers 23) and auxin transporter PIN8 (PIN-FORMED 8) are localized in the ER and regulate pollen growth in Arabidopsis. But how these two proteins regulate pollen growth remains to be studied. Here, we report that CHX23 and PIN8 act coordinately in regulating pollen growth. The chx23 mutant was reduced in pollen growth and normally shaped pollen grains, and complementation with CHX23 restored both pollen growth and normal pollen morphology. NAA treatments showed that CHX23 was crucial for pollen auxin homeostasis. The pin8 chx23 double mutant was decreased in pollen growth and normal pollen grains, indicating the joint effort of CHX23 and PIN8 in pollen growth. In vivo germination assay showed that CHX23 and PIN8 were involved in the early stage of pollen growth. CHX23 and PIN8 also function collaboratively in maintaining pollen auxin homeostasis. PIN8 depends on CHX23 in regulating pollen morphology and response to NAA treatments. CHX23 co-localized with PIN8, but there was no physical interaction. KCl and NaCl treatments showed that pollen growth of chx23 was reduced less than Col-0; pin8 chx23 was reduced less than chx23 and pin8. Together, CHX23 may regulate PIN8 function and hence pollen growth through controlling K+ and Na+ homeostasis mediated by its transport activity.

Wilson Disease With Novel Compound Heterozygote Mutations in the ATP7B Gene Presenting With Severe Diabetes.

OBJECTIVE: To determine the relationship between ATP7B mutations and diabetes in Wilson disease (WD). RESEARCH DESIGN AND METHODS: A total of 21 exons and exon-intron boundaries of ATP7B were identified by Sanger sequencing. RESULTS: Two novel compound heterozygous mutations (c.525 dupA/ Val176Serfs*28 and c.2930 C>T/ p.Thr977Met) were detected in ATP7B. After d-penicillamine (D-PCA) therapy, serum aminotransferase and ceruloplasmin levels in this patient were normalized and levels of HbA1c decreased. However, when the patient ceased to use D-PCA due to an itchy skin, serum levels of fasting blood glucose increased. Dimercaptosuccinic acid capsules were prescribed and memory recovered to some extent, which was accompanied by decreased insulin dosage for glucose control by 5 units. CONCLUSIONS: This is the first report of diabetes caused by WD.

A Targeted Multiple Antigenic Peptide Vaccine Augments the Immune Response to Self TGF-ß1 and Suppresses Ongoing Hepatic Fibrosis.

Transforming growth factor (TGF)-ß1 expression is induced upon liver injury, and plays a critical role in hepatic fibrosis. Antibodies against TGF-ß1 represent a novel approach in the treatment of hepatic fibrosis. However, TGF-ß1 is not a suitable antigen due to immunological tolerance. In the current study, we synthesized a multiple antigenic peptide (MAP) vaccine against the dominant B-cell epitope of TGF-ß1. The immunogenicity and potential therapeutic effects of this vaccine were examined using a rat model of hepatic fibrosis. Dominant B-cell epitopes of TGF-ß1 were identified using bioinformatic program. An MAP vaccine corresponding to the 90-98 amino acid domain of TGF-ß1 and containing four dendritic arms was synthesized using a 9-fluorenylmethoxycarbonyl solid phase method. Hepatic fibrosis which was induced in male Sprague-Dawley rats received a high-fat diet and ethanol (1.8 g/kg). Starting from the third week, rats were exposed to 40 % carbon tetrachloride (CCl4; 150 µl/100 g body weight twice weekly, initially 200 µl/100 g) treatment for a duration of 8 weeks. Rats received the MAP vaccine (100 µg) or Freund's adjuvant at weeks 1, 3, 5. A group of rats receiving the fibrosis-inducing regimen alone and a group of healthy rats (receiving an olive oil vehicle alone) were included as controls. At the conclusion of the experiment, serum titre of TGF-ß1 antibody was measured using ELISA and a standard liver functional test panel was examined. The extent of hepatic fibrosis was determined by measuring hydroxyproline content in the liver as well as hematoxylin-eosin (HE) and van Gieson (VG) staining. The expression of TGF-ß1 and alpha-smooth muscle actin (α-SMA) was examined using immunohistochemistry, and presented as positive staining cells. The MAP purity was >90 % upon reverse phase high-performance liquid chromatography, with apparent molecular weight at 4.77 kDa. Serum TGF-ß1 antibody titre was 1:256. The fibrosis-inducing treatment produced significant liver damage, as reflected by increases in liver functional test, HE and VG staining. The MAP vaccine attenuated such damage, as reflected by decreased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and hepatic hydroxyproline (116.78 ± 23.76 vs. 282.71 ± 136.94 IU/L; 319.78 ± 82.48 vs. 495.29 ± 137.13 IU/L; 2.02 ± 0.27 vs. 4.01 ± 0.52 µmol/L; 263.67 ± 41.18 vs. 439.14 ± 43.29 µg/g vs. in model rats, respectively; p < 0.01), as well as fibrosis extent by HE and VG staining. The MAP vaccine reduced TGF-ß1 and α-SMA expression in rats (0.325 ± 0.059 vs. 0.507 ± 0.044 IOD/area; 0.318 ± 0.058 vs. 0.489 ± 0.029 IOD/area vs. model rats, respectively; p < 0.05). The TGF-ß1 MAP vaccine could generate sufficient antibody that suppresses the development of hepatic fibrosis.

Effects of population size on synchronous display of female and male flowers and reproductive output in two monoecious Sagittaria species.

BACKGROUND: Flowering synchrony and floral sex ratio have the potential to influence the mating opportunities and reproductive success through female function. Here, we examine the variances in synchronous display of female and male function, ratio of male to female flowers per day and subsequently reproductive output in small populations of two monoecious plants, Sagittaria trifolia and Sagittaria graminea. METHODOLOGY/PRINCIPAL FINDING: We created plant populations of size 2, 4, 10 and 20 and recorded the daily number of blooming male and female flowers per plant to determine daily floral display, flowering synchrony index and ratio of male to female flowers per day. We also harvested the fruits, counted the seeds and calculated the number of fruits and seeds per flower to measure reproductive success through female function. There is less overlap in flowering time of female and male function in smaller populations than in larger populations. Most importantly, we found that male-biased floral sex ratio and imbalanced display period of female and male function for individual plant can lead to a population-size-dependent ratio of male to female flowers per day. Increasing ratio of male to female flowers per day was generally associated with a greater percentage of fruit production. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of flowering synchrony of female and male function and population-size-dependent ratio of male to female flowers per day for female reproductive success. This finding improves our understanding of a mechanism that reduces reproductive success in small populations.

Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x(L) protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase.

[The expression of mRNA and SDS-PAGE of L1210 cell strains and its cloned cells].

OBJECTIVE: To examine the expression difference of mRNA of L1210 cell strains and its cloned cells and discuss the methods for quality control of cell strains. METHODS: We used SDS-PAGE to observe the difference of protein and performed in situ hybridization to examine the expression of mRNA with the use of 6 cDNA probes that were marked by biotin. RESULTS: The number of protein bands of L1210 from Beijing Cancer Institute was 32. The number of protein bands of the two cloned cells L3E11 and L3F9 was 31. The 6 cDNA probes (p16, c-fos, c-jun, c-myc, p21, and p53 mRNA) were found to be existing in Beijing Cancer Institute L1210 and two different cloned cell strains. Expression of c-myc, c-fos, p53 mRNA could distinguish L3E11 and L3F9 cloned cells.