磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg⁻¹ and 0.96 µg L⁻¹ for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg⁻¹) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L⁻¹). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.     

Mechanism of endotoxin-induced reduction in the number of beta-adrenergic receptors in dog livers: role of phospholipase A

The role of phospholipase A on the endotoxin-induced reduction in the number of beta-adrenergic receptors in dog liver plasma membranes was investigated. The results show that digestion of control liver plasma membranes with exogenous phospholipase A2 (0.2 unit/200 micrograms protein) decreased the specific binding of (-)-[3H]dihydroalprenolol by 37.3% (P less than 0.01) and reduced the number of receptor sites by 31.7% (P less than 0.05). These decreases in the specific binding and the number of beta-adrenergic receptors were completely reversible by the addition of phosphatidylcholine (0.2 mM). Endotoxin administration (2 hr postendotoxin) decreased the specific binding by 36% (P less than 0.05) and reduced the number of beta-adrenergic receptors by 33% (P less than 0.05), and these decreases were completely reversible by the addition of 0.2 mM phosphatidylcholine. Digestion of control liver membranes with exogenous phospholipase A2 decreased phosphatidylcholine and phosphatidylethanolamine levels by 50.6 and 51.2%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine levels by 12- and 8.4-fold, respectively. Endotoxin administration decreased phosphatidylcholine and phosphatidylethanolamine contents by 21.4 and 23.8%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine contents by 2.1- and 1.4-fold, respectively. In addition, endotoxin administration increased endogenous phospholipase A activity by 73.5%. Based on these results, it is suggested that the decreases in the specific binding and the number of beta-adrenergic receptors in dog livers during endotoxic shock are a result of phospholipase A activation.     

Sphingosine kinase 1 regulates HMGB1 translocation by directly interacting with calcium/calmodulin protein kinase II-δ in sepsis-associated liver injury

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.Subject terms: Hepatotoxicity, Sepsis     

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.