Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp^−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp^−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp^−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.     

镉离子诱导BA/F3β细胞发生奇特的细胞凋亡

细胞凋亡一般都伴随有ＤＮＡ 片段化, 活性氧含量增加, 并能被过量的Ｂｃｌ２ 所抑制。以ＢＡ／Ｆ３β细胞为模型, 利用ＭＴＴ 检测、Ｈｏｃｈｅｓｔ 染色以及透射电镜检测等技术却发现, 镉离子虽然可以诱导该细胞凋亡, 但是这种凋亡没有ＤＮＡ 片段化, 也没有活性氧含量增加。此外, 过量Ｂｃｌ２ 对这种凋亡也没有保护作用。因此, 可以确认镉离子诱导ＢＡ／Ｆ３β细胞发生了奇特的细胞凋亡。     

Nitidine chloride inhibited the expression of S phase kinase-associated protein 2 in ovarian cancer cells

Nitidine chloride (NC) has been reported to exert its anti-tumor activity in various types of human cancers. However, the molecular mechanism of NC-mediated tumor suppressive function is largely unclear. In the current study, we used several approaches such as MTT, FACS, RT-PCR, Western blotting analysis, invasion assay, transfection, to explore the molecular basis of NC-triggered anti-cancer activity. We found that NC inhibited cell growth, induced cell apoptosis, caused cell cycle arrest in ovarian cancer cells. Emerging evidence has demonstrated that Skp2 plays an important oncogenic role in ovarian cancer. Therefore, we also explored whether NC exerts its biologic function via downregulation of Skp2 in ovarian cancer cells. We observed that NC significantly inhibited the expression of Skp2 in ovarian cancer cells. Notably, overexpression of Skp2 abrogated the anti-cancer activity induced by NC in ovarian cancer cells. Consistently, downregulation of Skp2 expression enhanced the sensitivity of ovarian cancer cells to NC treatment. Thus, inactivation of Skp2 by NC could be a novel strategy for the treatment of human ovarian cancer.     

Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.     

Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.     

Mitochondrial oxidative metabolism during respiratory infection in riboflavin deficient mice

Studies in children and mice have shown that respiratory infection alters riboflavin metabolism, resulting in increased urinary loss of this vitamin. This could be due to mobilization of riboflavin from the liver to blood because liver Flavin adenine dinucleotide (FAD) levels were lowered in the mice during infection. To understand the functional implications of lowered hepatic FAD levels during respiratory infection, flavoprotein functions such as oxidative phosphorylation and β-oxidation of the liver mitochondria were examined during infection in mice. Weanling mice were fed either riboflavin-restricted or control diet for 18 days and then injected with a sublethal dose of Klebsiella pneumoniae. During infection, the state 3 respiratory rate with palmitoyl-L-carnitine and glutamate were significantly lowered (27–29%) in the riboflavin-restricted group, whereas in the control group 10% reduction was observed with palmitoyl-L-carnitine as substrate. A 22% reduction in the respiratory control ratio with palmitoyl-L-carnitine as substrate was observed during infection in the riboflavin-restricted group. The β-oxidation of palmitoyl-L-carnitine was significantly lowered (29%) in the riboflavin-restricted infected group. The results of the study suggest that the effects of infection on vital physiologic functions were more pronounced in the riboflavin-restricted mice than in the control mice. © Elsevier Science Inc. 1999     

Kinin-mediated inflammation in neurodegenerative disorders

The mediatory role of kinins in both acute and chronic inflammation within nervous tissues has been widely described. Bradykinin, the major representative of these bioactive peptides, is one of a few mediators of inflammation that directly stimulates afferent nerves due to the broad expression of specific kinin receptors in cell types in these tissues. Moreover, kinins may be delivered to a site of injury not only after their production at the endothelium surface but also following their local production through the enzymatic degradation of kininogens at the surface of nerve cells. A strong correlation between inflammatory processes and neurodegeneration has been established. The activation of nerve cells, particularly microglia, in response to injury, trauma or infection initiates a number of reactions in the neuronal neighborhood that can lead to cell death after the prolonged action of inflammatory substances. In recent years, there has been a growing interest in the effects of kinins on neuronal destruction. In these studies, the overexpression of proteins involved in kinin generation or of kinin receptors has been observed in several neurologic disorders including neurodegenerative diseases such Alzheimer's disease and multiple sclerosis as well as disorders associated with a deficiency in cell communication such as epilepsy. This review is focused on recent findings that provide reliable evidence of the mediatory role of kinins in the inflammatory responses associated with different neurological disorders. A deeper understanding of the role of kinins in neurodegenerative diseases is likely to promote the future development of new therapeutic strategies for the control of these disorders. An example of this could be the prospective use of kinin receptor antagonists.