Prognostic alternative splicing regulatory network of splicing events in acute myeloid leukemia patients based on SpliceSeq data from 136 cases.

This study aimed to create prognostic signatures to predict AML patients' survival using alternative splicing (AS) events. The AS data, RNA sequencing data, and the survival statistics of 136 AML patients were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Total 34,984 AS events generated from 8,656 genes, 2,583 of which were survival-associated AS events, were identified using univariate Cox regression. The prognostic models constructed using independent survival-associated AS events revealed that low-risk splicing better predicted patients' survival. ROC analysis indicated that the predictive efficacy of the alternate terminator model was best in the area under the curve at 0.781. Enrichment analysis revealed several important genes (TP53, BCL2, AURKB, PPP2R1B, FOS, and BIRC5) and pathways, such as the protein processing pathway in the endoplasmic reticulum, RNA transport pathway, and HTLV-I infection pathway. The splicing network of splicing events and factors revealed interesting interactions, such as the positive correlation between HNRNPH3 and CALHM2-13010-AT, which may indicate the potential splicing regulatory mechanism. Taken together, survival-associated splicing events and the prognostic signatures for predicting survival can help provide an overview of splicing in AML patients and facilitate clinical practice. The splicing regulatory network may improve the understanding of spliceosomes in AML.

LncRNA CASC11 promotes the development of esophageal carcinoma by regulating KLF6.

OBJECTIVE: The aim of this study was to investigate the expression level of long non-coding RNA (lncRNA) CASC11 in esophageal carcinoma (ECa), and to further explore its relationship with clinical progression, pathological parameters, and prognosis of ECa patients. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine the level of lncRNA CASC11 in 45 pairs of ECa tissues and adjacent normal tissues. The relationship between the lncRNA CASC11 level and clinical progression, pathological parameters, and prognosis of ECa patients was analyzed. Meanwhile, the level of lncRNA CASC11 in the ECa cell lines was verified by qPCR as well. In addition, lncRNA CASC11 knockdown model was constructed using lentiviral transfection in ECa cell lines. Subsequently, the cell counting kit-8 (CCK8), colony formation assay, and flow cytometry were used to explore the effect of lncRNA CASC11 on the biological functions of the ECa cells. Finally, the Western Blot and the recovery experiments were used to explore the potential mechanism. RESULTS: In this work, the qPCR results showed that the expression level of lncRNA CASC11 in the ECa tissues was remarkably higher than that of the adjacent normal tissues, and the difference was statistically significant (p<0.05). Compared with patients with a low level of lncRNA CASC11, the pathological stage of patients with high expression was significantly higher, while the overall survival rate was lower (p<0.05). Compared with negative control (NC) group, the proliferation ability of the cells in the lncRNA CASC11 knockdown group CASC11 significantly decreased, whereas cell apoptosis remarkably increased (p<0.05). The Western Blot results revealed that protein expression of KLF6 was remarkably up-regulated after lncRNA CASC11 knockdown. In addition, the recovery experiments found that lncRNA CASC11 and KLF6 had mutual regulation, thereby promoting the malignant progression of ECa. CONCLUSIONS: LncRNA CASC11 expression was remarkably up-regulated in ECa, which was associated with the pathological stage and poor prognosis of ECa. In addition, lncRNA CASC11 could promote the malignant progression of ECa by mutual regulation of KLF6.

Effect of exogenous gibberellin on reserve accumulation during the seed filling stage of oilseed rape.

Exogenous gibberellins (GAs) are widely applied to increase crop yields, with knowledge about the physiological functioning and biochemistry mechanisms of these phytohormones improving; however, information remains limited about the effect of GAs on seed filling. In this study, the siliques (containing the seeds) of oilseed rape (Brassica napus L.) were treated with GA3 at 3 stages of seed filling. We confirmed that GA3 regulates the deposition of storage reserves in developing seeds. The percentage of crude fat in the seeds increased during the early stage, but remained stable during the middle and late stages. In comparison, the percentage of total protein decreased during the early and middle stages, but significantly increased during the late stage. In addition, Q-PCR was employed to analyze the expression level of related genes in response to GA3. It was found that the expression of WRI and ABI3 transcription factors corresponded to crude fat content and total protein content, respectively. The expression of storage reserve related genes DGAT, MCAT, SUC2, and GPT was consistent with crude fat content, whereas the expression of Napin corresponded to total protein content. The results of this study indicate that exogenous GA3 has a different effect on storage reserve deposition in seed during different stages of seed filling, and the effect might be achieved via changing the expression of related genes.

Extracellular signal-regulated protein kinase in human intractable epilepsy.

Extracellular signal-regulated kinases (ERK) such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the central nervous system under physiological and pathological conditions such as ischemia and epilepsy. Our aim is to investigate ERK1, ERK2, and phosphorylated ERK (p-ERK) (Thr202/Tyr 204) expression in the temporal lobe of patients with intractable epilepsy (IE) and to explore its possible role of ERK in it. Tissue samples from temporal neocortices of 40 patients who had surgery for IE were used to detect ERK1, ERK2, and p-ERK (Thr 202/Tyr 204) expression through immunohistochemistry and western blot. We compared these tissues against 17 histological normal temporal lobes from head-trauma patients. ERK1, ERK2, and p-ERK in IE were significantly higher than those in the controls. They were mainly expressed in the cytoplasm of neurons and glial cells. There was also increased detection of p-ERK in the gliotic cortex of IE compared with the non-gliotic cortex. These findings were consistently observed in western blot and immunohistochemistry techniques. ERK expression in patients with IE was significantly increased compared with the controls. This suggested a probable role of ERK in the pathogenesis of IE.

Microtubule associated protein tau binds to double-stranded but not single-stranded DNA.

Tau, a major microtubule-associated protein of the neuron, which is known to promote the assembly of and to stabilize microtubules, has also been seen associated with chromatin in neuronal cell lines, but its role in this subcellular compartment is still unknown. In this study, the binding of tau to DNA was investigated using the electrophoretic mobility shift assay. Using polynucleotide as probe, we found that tau bound to double-stranded but not to single-stranded DNA. Formation of tau-polynucleotide complex was disrupted by alkaline pH and a high concentration of NaCl, but was not affected by dithiothreitol. Electron microscopy revealed that the protein associated with the nucleic acid in a necklacelike manner. DNA-cellulose chromatography and radioimmunodot-blot analyses showed that calf thymus histones VI-S, VII-S and VIII-S could replace both recombinant human brain tau352 (tau-23) and tau441 (tau-40) from DNA. Thus, tau appears to bind to DNA reversibly in the presence of histones.

Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis.

vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral eye development. At early neurula stages, while Xpax2 starts to be expressed within the eye field, both Xvax2 and Xvax1b are exclusively activated in the presumptive ventral telencephalon. Since midneurula stages, Xvax2 and Xvax1b are also transcribed in the medial aspect of the eye field. At tailbud and tadpole stages, Xvax2, Xvax1b and Xpax2 expression overlaps in the optic stalk and nerve and in the optic disk, while Xvax2 and Xvax1b also display specific activation domains in the ventral retina as well as in the ventral telencephalon and diencephalon. Finally, during metamorphosis a high level of both Xvax2 and Xvax1b transcription is maintained in the optic chiasm. In addition, Xvax1b is transcribed in the ventral hypothalamus and in the hypophysis, whereas a strong Xvax2 expression is retained in the ventral portion of the mature retina.

Homeobox genes in the genetic control of eye development.

Vertebrate eye formation is a complex process which involves early specification of the prospective eye territory, induction events, patterning along the polarity axes and regional specification, to bring about the proper morphogenetic movements, cell proliferation, cell differentiation and neural connections allowing visual function. The molecular machinery underlying such complex developmental events is presently under an intense research scrutiny and many associated genetic factors have been isolated and characterized. These studies produced striking knowledge in the field, especially with respect to uncovering the role of key genes and their possible evolutionary conservation. Presently, a major task is to define the complex interactions connecting the multiplicity of molecular players that regulate eye development. We recently identified two homeobox genes, Xrx1 and Xvax2, and studied their function by using the Xenopus embryo as a developmental model system. Xrx1 and Xvax2 control key aspects of eye development. In particular, Xrx1 appears to play a role in the early specification of anterior neural regions fated to give rise to retina and forebrain structures, and in promoting cell proliferation within these territories. On the other hand, Xvax2 is involved in regulating the eye proximo-distal and/or dorsoventral polarity, and the morphogenetic movements taking place during formation of the optic stalk and cup. Here we review the experimental results addressing the roles of Xrx1 and Xvax2 and their vertebrate orthologues, and discuss their relationship with other molecules also playing a related function in eye development.

Effect of human neuronal tau on denaturation and reactivation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase.

Human neuronal tau-40 (htau-40) has been used to study denaturation and renaturation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). Inactivation of GAPDH incubated with tau was more distinguishably detected than that of control GAPDH during thermal and guanidine hydrochloride (GdnHCl) denaturation. However, tau did not influence the activity of GAPDH at room temperature or in solution without GdnHCl. A marked change in both the emission intensity and emission maximum of the intrinsic fluorescence at 335 nm of GAPDH with tau was observed when GdnHCl concentration was 0.8 M, but that of the control without tau occurred in 1.2 M GdnHCl. The first-order rate of the decrease in the fluorescence intensity of the enzyme with tau was approximately twice as great as that of GAPDH without tau. Kinetics of inactivation of GAPDH with tau in 0.2 M GdnHCl was a monophasic procedure, instead of the biphasic procedure followed by the control, as described before [He, Zhao, Yan and Li (1993) Biochim. Biophys. Acta 1163, 315-320]. Similar results were obtained when the enzyme was thermally denatured at 45 degrees C. It revealed that tau bound to the denatured GAPDH but not the native molecule. On the other hand, tau suppressed refolding and reactivation of GAPDH when this enzyme was reactivated by dilution of GdnHCl solution. Furthermore, tau improved the aggregation of the non-native GAPDH in solutions. It suggested that tau acted in an anti-chaperone-like manner towards GAPDH in vitro. However, tau lost that function when it was aggregated or phosphorylated by neuronal cdc2-like protein kinase. It showed that tau's anti-chaperone-like function depended on its native conformation.

Estimation of the optimal concentrations of residual sugar and cell growth rate for a fed-batch culture of Saccharomyces cerevisiae.

Estimation of the optimal concentrations of residual sugar in medium for a fed-batch culture of Baker's yeast has been studied and practiced. The concentrations, however, depended on different species and targets of the biomass, which was expected to be made. Kinetic changes of the residual phosphate salt in the medium conformed to a logarithmic process until the fourth hour during an 11-h culture. The parabolic method (see ref. 9 later in article) might be qualified to maintain the concentrations of residual sugar around 0.15 g/L. It was demonstrated that cell growth followed a sigmoid process during a fed-batch culture, because the cells consumed the nutrient with two metabolic pathways, one was for cell conversion and another was for non-cell conversion. With the parabolic method, we can estimate kinetics of cell growth and cell growth rate during the culture.

Formation of fluorophore of yeast D-glyceraldehyde-3-phosphate dehydrogenase in the solution of potassium iodide.

A new fluorescence property of yeast D-glyceraldehyde-3-phosphate dehydrogenase was detected after it had been incubated in KI solution. The fluorophore formed in Baker's yeast GAPDH fluoresced at 383 nm by excitation at 336 nm. The formation of the fluorophore depended on NAD+. Modification of Cys-149 with iodoacetic acid influenced some properties of the fluorescence. It appears that the fluorophore may be situated at or near the active site.

Inactivation and conformation changes of the glycated and non-glycated D-glyceraldehyde-3-phosphate dehydrogenase during guanidine-HCl denaturation.

The glycated D-glyceraldehyde-3-phosphate dehydrogenases have been isolated from rabbit muscle and erythrocytes (He et al. (1995) Biochem. J. 309, 133-139). The circular dichroism spectrum in the near-ultraviolet of gGAPDH was different from that of GAPDH. Changes in intrinsic protein fluorescence and in the 410 nm fluorescence of the NAD derivatives introduced at the active sites of both the glycated and non-glycated GAPDH from rabbit were compared on inactivation during denaturation in GuHCl. Complete inactivation for the non-glycated enzyme occurred in 0.5 M GuHCl solution, however, that for the glycated enzyme occurred in the 0.35 M solution. The kinetic inactivation of gGAPDH was a biphasic process (the fast and slow phases). The fast phase for gGAPDH was faster than that of GAPDH. The kinetic exposure of the fluorescent NAD derivatives at the active sites of both enzymes was also biphasic with fast phase rates which approach those of the inactivation. It appears that glycation of the enzyme may disturb the spatial geometry of the functional groups responsible for the catalytic mechanism and affect the activity.

Some fluorescent properties of non-enzymic transamination and measurement of the relative molecular mass of protein.

A procedure for transaminating proteins and removing the transaminated N-terminal residue has been used for studying structure-function relationship of protein (Dixon and Fields 1972, Meth. Enzymol. 25, 409-419). We show that it is convenient for measuring the relative molecular masses of proteins by measuring the glycine formed from glyoxylate during such transamination. Quinoxaline derivatives have been synthesized by the reaction of 2-oxo acids from amino acids reacting with o-phenylenediamine. 2-Oxo acids transaminated from amino acids fluoresced at around 405 nm, depending on the nature of residual side groups. The emission maximum of 3-benzyl-2-hydroxy-quinoxaline from the reaction of o-phenylenediamine with phenylpyruvate was at 363 nm. The fluorescent derivatives have been used to study conformational changes of peptides and to detect whether or not N-termini of proteins were blocked.

Isolation and some properties of glycated D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle.

Glycated D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from rabbit muscle and human erythrocytes have been investigated. The specific activity of the non-glycated GAPDH from rabbit muscle is approx. 180 units. (One unit is defined as the specific activity required to convert 1 microM of substrate/min per mg of enzyme.) The activity of the glycated enzyme, consisting of two sugars per tetramer, is lower than that of the non-glycated GAPDH. Non-enzymic transamination of the N-termini of glycated GAPDH (gGAPDH) indicates that they are not blocked by glycation. The rate of modification of thiols (Cys-149) with 5,5'-dithiobis-(2-nitrobenzoic acid) was greater for the glycated than the non-glycated enzymes. The rate of modification of amino groups of Lys residues of gGAPDH with o-phthalaldehyde was greater for the non-glycated enzyme. In 0.18 M guanidine-HC1 solution, the emission intensity at 410 nm of a fluorescent NAD+ derivative introduced into the active site decreased to 80%, whereas that of gGAPDH decreased to 50%. This suggests that the glycated sites are near the active site; glycation of the enzyme leads to a change of the microenvironment of Cys-149, alters the conformation of the active site and decreases the activity.

Changes in RNA of Saccharomyces cerevisiae during thermal cellular dehydration.

Changes in RNA of Baker's yeast have been studied during a thermal dehydration, through which the cellular moisture decreased from 70% to 9%, followed by a mortality of cells being less than 8%, and by consumption of the cellular glycogen. Simultaneously, the RNA of 4.2s and 5.8s increase, however, the RNA of 12.0s and 18.0s decrease during the thermal cellular dehydration. The contents of the low molecular mass RNA may not be affected by incubation of chloromycetin and penicillin in media during the fed-batch culture. It suggests that RNA play a part during the cellular thermal dehydration.

The principle of parabolic feed for a fed-batch culture of baker's yeast.

A parabolic nutrient supplementation has been derived by the specific growth rate, sugar conversion rate, and Pasteur effect, followed by high qualitative and quantitative biomass production with the lowest sugar consumption. The method produced about 95% cells in the G1 phase that were more resistant to drying and aging. These features are particularly important in the process of making dry yeast. It appears that the parabolic feed method may be used to species culture that show the Pasteur effect or produce byproducts from sugar. This may be because the supplementation is in conformity with reproducible kinetic growth during the fed-batch culture.

An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp. during guanidine denaturation.

Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp. (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions. The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues. The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH. The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C. The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes. The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution. Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition. It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.