Operation of the glucose-fatty acid cycle after glycogenolysis induced by treatment with nicotinic acid.

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work (J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.     

A model for dynamics of cell division cycle in onion roots

Summary The study of the cell division cycle by means of caffeine labelling inAllium roots, at 15° C, employing intact root and decapitated roots at several levels (0.5, 1.0, 1.5, 2.0, and 2.5 mm) has shown that the number of cycles developed by the cells is constant at each meristem level. This number and the durations of the cycles are not affected by the decapitation. It is suggested that the cell cycle is controlled in the meristematic cells by an intracellular programme which would be developed throughout the meristem.However, the larger the region decapitated is, the more decreases the growth rate of the roots. The removal of the root cap (about 0.5 mm) did not modify the rate of root growth, although it blocked the geotropic response. The quiescent center is proposed as a source of auxin controlling cell elongation.     

Protein kinase from Mucor rouxii

Summary Cyclic AMP binding to Mucor rouxii protein kinase holoenzyme and free regulatory subunit was measured by the classical membrane filtration technique and by equilibrium dialysis. The results obtained demonstrate that the filtration method can be used without loss of any cyclic AMP binding site. Both methods unambiguously demonstrate that the number of molecules of cyclic AMP bound to the holoenzyme are half of those bound to the regulatory subunit. This result suggests that unshielding of new cyclic AMP binding sites occurs upon dissociation of the ternary complex holoenzyme-cyclic AMP.     

Telomere length alterations in microsatellite stable colorectal cancer and association with the immune response

Telomeres are repetitive sequences (TTAGGG) located at the end of chromosomes. Telomeres progressively shorten with each cell replication cycle, ultimately leading to chromosomal instability and loss of cell viability. Telomere length anomaly appears to be one of the earliest and most prevalent genetic alterations in malignant transformation. Here we aim to estimate telomere length from whole-exome sequencing data in colon tumors and normal colonic mucosa, and to analyze the potential association of telomere length with clinical factors and gene expression in colon cancer.Reads containing at least five repetitions of the telomere sequence (TTAGGG) were extracted from the raw sequences of 42 adjacent normal-tumor paired samples. The number of reads from the tumor sample was normalized to build the Tumor Telomere Length Ratio (TTLR), considered an estimation of telomere length change in the tumor compared to the paired normal tissue. We evaluated the associations between TTLR and clinical factors, gene expression and copy number (CN) aberrations measured in the same tumor samples.Colon tumors showed significantly shorter telomeres than their paired normal samples. No significant association was observed between TTLR and gender, age, tumor location, prognosis, stromal infiltration or molecular subtypes. The functional gene set enrichment analysis showed pathways related to immune response significantly associated with TLLR.By extracting a relative measure of telomere length from whole-exome sequencing data, we have assessed that colon tumor cells predominantly shorten telomeres, and this alteration is associated with expression changes in genes related to immune response and inflammation in tumor cells.     

Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23

Key message

Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle.

Abstract

The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

     

Role of genetic modifiers in an orthologous rat model of ARPKD

Human data and animal models of autosomal recessive polycystic kidney disease (ARPKD) suggest that genetic factors modulate the onset and severity of the disease. We report here for the first time that ARPKD susceptibility is attenuated by introgressing the mutated Pkhd1 disease allele from the polycystic kidney (PCK) rat onto the FHH (Fawn-Hooded Hypertensive) genetic background. Compared with PCK, the FHH.Pkhd1 strain had significantly decreased renal cyst formation that coincided with a threefold reduction in mean kidney weights. Further analysis revealed that the FHH. Pkhd1 is protected from increased blood pressure as well as elevated plasma creatinine and blood urea nitrogen levels. On the other hand, liver weight and biliary cystogenesis revealed no differences between PCK and FHH.Pkdh1, indicating that genes within the FHH genetic background prevent the development of renal, but not hepatic, manifestations of ARPKD. Microarray expression analysis of kidneys from 30-day-old PCK rats revealed increased expression of genes previously identified in PKD renal expression profiles, such as inflammatory response, extracellular matrix synthesis, and cell proliferation genes among others, whereas the FHH.Pkhd1 did not show activation of these common markers of disease. This newly developed strain can serve as a tool to map modifier genes for renal disease in ARPKD and provides further insight into disease variability and pathophysiology.     

Polymorphisms affecting micro-RNA regulation and associated with the risk of dietary-related cancers: a review from the literature and new evidence for a functional role of rs17281995 (CD86) and rs1051690 (INSR), previously associated with colorectal cancer

In this review, we focus on the genetic variations (single nucleotide polymorphisms, SNPs) known to occur in microRNAs and in their binding sites and the susceptibility to cancers of the gastro-intestinal (GI) tract in humans. Since the sequence complementarity and the thermodynamics of binding play an essential role in the interaction of miRNA with its target mRNA, sequence variations in the miRNA-binding seed regions or in miRNA genes (either within pre-, pri-, or mature miRNA regions) should reinforce, weaken, or disrupt the miRNA-mRNA interaction and affect the expression of mRNA targets. Indirect evidences supporting these hypotheses are reported in the literature, essentially coming from case-control association studies. Several studies have been published on the association between miR-SNPs or SNPs within their binding sites and the risk of oesophageal, gastric, or colorectal cancer. Unfortunately, functional studies are lacking. Besides reviewing the available literature, we present here for the first time two SNPs (rs17281995 in CD86 and rs1051690 in INSR) previously associated with the risk of CRC in a Czech population are also associated with the risk in a Spanish population. Moreover, we show for the first time that both these alleles regulate differentially the amount of a reporter gene (luciferase) in an in vitro assay on HeLa cells. These findings suggest that both these SNPs may have a functional role in regulating the expression of CD-86 and INSR proteins acting at the level of the 3'UTR. More functional studies are needed in order to better understand the role of polymorphic regulatory sequences at the 3'UTR of genes.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.