Evaluation of the mature grain phytase candidate <Emphasis Type="Italic">HvPAPhy_a</Emphasis> gene in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) using CRISPR/Cas9 and TALENs

In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA. Additionally, the analysis of the HvPAPhy_a promoter region containing the GCN4/Skn1/RY motif highlights its importance for HvPAPhy_a expression as the MGPA in grains of plant lines with mutations within this motif is significantly reduced. Interestingly, lines with deletions located downstream of the motif show even lower MGPA levels, indicating that the GCN4/SKn1/RY motif is not the only element responsible for the level of PAPhy_a expression during grain maturation. Mutant grains with very low MPGA showed delayed germination as compared to grains of wild type barley. As grains with high levels of preformed phytases would provide more readily available phosphorous needed for a fast germination, this indicates that faster germination may be implicated in the positive selection of the ancient PAPhy gene duplication that lead to the creation of the PAPhy_a gene.     

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.Subject terms: Biochemistry, Cell biology     

iTRAQ-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated with progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.     

Changes in adrenocortical monooxygenase activities in alloxan-diabetic rabbits

Studies were carried out to determine if diabetes mellitus influenced the activities of adrenal steroidogenic enzymes. Adult male rabbits were made diabetic by an i.v. infusion of alloxan (100 mg/kg) and were killed 1 or 2 months later. Mitochondrial cytochrome P-450 concentrations were not affected by diabetes but steroid 11 beta-hydroxylase activity was greater in the diabetics than in controls after both 1 and 2 months. The type I spectral change produced by 11-deoxycorticosterone, the substrate for 11 beta-hydroxylation, was also greater in mitochondria from diabetics. By contrast, mitochondrial cholesterol side-chain cleavage activity was similar in controls and diabetics. Microsomal cytochrome P-450 concentrations were unaffected by diabetes but 21-hydroxylase activity was significantly lower in adrenal microsomes from diabetics than from controls. The results indicate that alloxan-induced diabetes alters adrenocortical steroid metabolism which may contribute to changes in the pattern of steroid secretion noted by other investigators.     

Divergence of growth hormone actions on hepatic drug metabolism in the presence and absence of the pituitary gland.

Experiments were carried out to compare the effects of growth hormone on hepatic drug oxidation in normal and hypophysectomized rats. Administration of growth hormone to normal male rats lowered hepatic microsomal cytochrome P-450 content and decreased the rates of ethylmorphine n-demethylation and aniline hydroxylation. These effects were fully manifested in orchiectomized or adrenalectomized males, excluding a dependence upon endogenous steroids. Growth hormone was without effect on hepatic drug metabolism or cytochrome P-450 content in normal female rats. In contrast to its actions in animals with intact pituitary glands, administration of growth hormone to hypophysectomized rats of either sex increased the rate of ethylmorphine metabolism. Furthermore, in both males and females, aniline hydroxylation and microsomal cytochrome P-450 content were unaffected by growth hormone in the absence of the pituitary gland. Prolactin administration did not affect hypophysectomized or in normal rats of either sex. The results indicate that the nature of growth hormone actions on hepatic drug oxidation is pituitary-dependent and probably intertwined with the effects of other hormones. Furthermore, the direct physiological effects of growth hormone on hepatic mixed function oxidases seem to depend upon the substrate employed.     

A highly specific and sensitive determination of gamma-aminobutyric acid by gas chromatography mass spectrometry

A procedure for the identification and quantification of picomole quantities of gamma-aminobutyric acid in tissue samples is given. This procedure combines the chemical specificity of dinitrophenylation with that of gas chromatography mass spectrometry to eliminate the interferences encountered with other direct derivatization procedures. Only a limited number of dinitrophenyl amino acid ethyl esters and some fatty ethyl esters are detected in the solution used for analysis. Identification is based on retention time and on the relative abundances of the three major ion fragments of the gamma-aminobutyric acid derivative. Quantitation is accomplished using isotope dilution techniques with [2H2]gamma-aminobutyric acid as an internal standard. The procedure has been successfully applied to samples of human cerebrospinal fluid and to extracts of ganglia from the mollusc, Aplysia californica.     

Regional distribution of microsomal drug and steroid metabolism in the guinea pig adrenal cortex

The regional distribution of steroid and drug metabolism was studied in intact cells and microsomal fractions obtained from the chromatically distinct inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Cells isolated from the outer cortical zone produced far more cortisol than cells from the inner zone and cortisol production was stimulated by adrenocorticotropic hormone only in cells from the outer zone. Among the factors which may contribute to the greater cortisol production by the outer zone are a higher rate of 17 alpha-hydroxylation and ratio of 17 alpha- to 21-hydroxylase activities in that zone, both of which favor cortisol synthesis. In contrast, steroid 21-hydroxylase activity was far greater than 17 alpha-hydroxylase activity in microsomes obtained from the inner zone of the adrenal cortex. Microsomal metabolism of various xenobiotics such as benzo(a)pyrene and ethylmorphine proceeded far more rapidly in the inner than outer cortical zone. The zonal differences in metabolism appeared to result in part from differences in the ability of xenobiotics to interact with microsomal cytochromes P-450 in the two zones. The results indicate that the inner zone has a minor role in cortisol production by the adrenal cortex, but its involvement in the production of other steroids cannot be excluded. In contrast, the inner zone appears to have the major role in the metabolism of at least some xenobiotics which may account for its greater vulnerability to the toxic effects of chemicals requiring metabolic activation.