Identification of QTLs Associated with Conversion of Sucrose to Hexose in Mature Fruit of Japanese Pear

Sweetness is the most important trait for fruit breeding and is fundamentally determined by both total and individual sugar contents. We analyzed the contents of sucrose, fructose, glucose, and sorbitol in mature fruit in an F₁ population derived from crossing modern Japanese pear cultivar ‘Akizuki’ and breeding line ‘373-55’. A genetic linkage map was constructed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP). We identified two regions associated with individual sugar contents on linkage group (LG) 1 and LG 7. The percentages of the variance in sucrose, fructose, and glucose explained by the quantitative trait loci (QTLs) were 26.6, 15.9, and 18.5%, respectively, for the region on LG 1, and 22.2, 20.0, and 9.5%, respectively, for the region on LG 7. In both regions, genotypes associated with increases in sucrose were associated with decreases in both fructose and glucose. The 1.5-LOD support intervals of the QTLs on LGs 1 and 7 include SSRs within the regions flanking acid invertase genes PPAIV3 and PPAIV1, respectively. Because acid invertase is a key enzyme in the conversion of sucrose to hexose, these are promising candidates for genes underlying those QTLs and controlling individual sugar contents. We also found one region on LG 11 that explained 21.4% of the variation in total sugar content but was not significantly associated with variation for individual sugars. The information obtained in this study will accelerate research and breeding programs to improve fruit sweetness.     

Genome sequencing and analysis of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’

To gain genetic insights into the early-flowering phenotype of ornamental cherry, also known as sakura, we determined the genome sequences of two early-flowering cherry (Cerasus × kanzakura) varieties, ‘Kawazu-zakura’ and ‘Atami-zakura’. Because the two varieties are interspecific hybrids, likely derived from crosses between Cerasus campanulata (early-flowering species) and Cerasus speciosa, we employed the haplotype-resolved sequence assembly strategy. Genome sequence reads obtained from each variety by single-molecule real-time sequencing (SMRT) were split into two subsets, based on the genome sequence information of the two probable ancestors, and assembled to obtain haplotype-phased genome sequences. The resultant genome assembly of ‘Kawazu-zakura’ spanned 519.8 Mb with 1,544 contigs and an N50 value of 1,220.5 kb, while that of ‘Atami-zakura’ totalled 509.6 Mb with 2,180 contigs and an N50 value of 709.1 kb. A total of 72,702 and 69,528 potential protein-coding genes were predicted in the genome assemblies of ‘Kawazu-zakura’ and ‘Atami-zakura’, respectively. Gene clustering analysis identified 2,634 clusters uniquely presented in the C. campanulata haplotype sequences, which might contribute to its early-flowering phenotype. Genome sequences determined in this study provide fundamental information for elucidating the molecular and genetic mechanisms underlying the early-flowering phenotype of ornamental cherry tree varieties and their relatives.     

Association between the blood concentrations of ammonia and carnitine/amino acid of schizophrenic patients treated with valproic acid

Background

Administration of valproic acid (VPA) is complicated with approximately 0.9% of patients developing hyperammonemia, but the pathogenesis of this adverse effect remains to be clarified. The aim of the present study was to search for mechanisms associated with VPA-induced hyperammonemia in the light of changes in serum amino acids concentrations associated with the urea cycle of schizophrenic patients.

Method

Blood samples (10 mL) were obtained from 37 schizophrenic patients receiving VPA for the prevention of violent behaviors in the morning after overnight fast. Blood concentrations of ammonia, VPA, free carnitine, acyl-carnitine, and 40 amino acids including glutamate and citrulline were measured for each patient. Univariate and multivariate regression analyses were performed to identify amino acids or concomitantly administered drugs that were associated with variability in the blood concentrations of ammonia.

Result

The blood ammonia level was positively correlated with the serum glutamate concentration (r = 0.44, p < 0.01) but negatively correlated with glutamine (r = ?0.41, p = 0.01), citrulline (r = ?0.42, p = 0.01), and glycine concentrations (r = ?0.54, p < 0.01). It was also revealed that the concomitant administration of the mood stabilizers (p = 0.04) risperidone (p = 0.03) and blonanserin (p < 0.01) was positively associated with the elevation of the blood ammonia level.

Conclusion

We hypothisized that VPA would elevate the blood ammonia level of schizophrenic patients. The observed changes in serum amino acids are compatible with urea cycle dysfunction, possibly due to reduced carbamoyl-phosphate synthase 1 (CPS1) activity. We conclude that VPA should be prudently prescribed to schizophrenic patients, particularly those receiving mood stabilizers or certain antipsychotics.

     

Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein

Background

The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes.

Methods and Findings

We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions.

Conclusions

Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.     

Structure-activity study of estrogenic agonists bearing dicarba-closo-dodecaborane. Effect of geometry and separation distance of hydroxyl groups at the ends of molecules

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have investigated structure-activity relations based on the structure of the potent estrogenic agonist, 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-d odecaborane, which we have previously reported. The geometry and separation distance of the phenolic and alcoholic hydroxyl groups play a critical role in the appearance of biological activity.     

Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification

MOTIVATION: Genes are often characterized dichotomously as either housekeeping or single-tissue specific. We conjectured that crucial functional information resides in genes with midrange profiles of expression. RESULTS: To obtain such novel information genome-wide, we have determined the mRNA expression levels for one of the largest hitherto analyzed set of 62 839 probesets in 12 representative normal human tissues. Indeed, when using a newly defined graded tissue specificity index tau, valued between 0 for housekeeping genes and 1 for tissue-specific genes, genes with midrange profiles having 0.15< tau<0.85 were found to constitute >50% of all expression patterns. We developed a binary classification, indicating for every gene the I(B) tissues in which it is overly expressed, and the 12-I(B) tissues in which it shows low expression. The 85 dominant midrange patterns with I(B)=2-11 were found to be bimodally distributed, and to contribute most significantly to the definition of tissue specification dendrograms. Our analyses provide a novel route to infer expression profiles for presumed ancestral nodes in the tissue dendrogram. Such definition has uncovered an unsuspected correlation, whereby de novo enhancement and diminution of gene expression go hand in hand. These findings highlight the importance of gene suppression events, with implications to the course of tissue specification in ontogeny and phylogeny. AVAILABILITY: All data and analyses are publically available at the GeneNote website, http://genecards.weizmann.ac.il/genenote/ and, GEO accession GSE803. CONTACT: doron.lancet@weizmann.ac.il SUPPLEMENTARY INFORMATION: Four tables available at the above site.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.