Trade-offs of lipid remodeling in a marine predator-prey interaction in response to phosphorus limitation.

Phosphorus (P) is a key nutrient limiting bacterial growth and primary production in the oceans. Unsurprisingly, marine microbes have evolved sophisticated strategies to adapt to P limitation, one of which involves the remodeling of membrane lipids by replacing phospholipids with non-P-containing surrogate lipids. This strategy is adopted by both cosmopolitan marine phytoplankton and heterotrophic bacteria and serves to reduce the cellular P quota. However, little, if anything, is known of the biological consequences of lipid remodeling. Here, using the marine bacterium Phaeobacter sp. MED193 and the ciliate Uronema marinum as a model, we sought to assess the effect of remodeling on bacteria-protist interactions. We discovered an important trade-off between either escape from ingestion or resistance to digestion. Thus, Phaeobacter grown under P-replete conditions was readily ingested by Uronema, but not easily digested, supporting only limited predator growth. In contrast, following membrane lipid remodeling in response to P depletion, Phaeobacter was less likely to be captured by Uronema, thanks to the reduced expression of mannosylated glycoconjugates. However, once ingested, membrane-remodeled cells were unable to prevent phagosome acidification, became more susceptible to digestion, and, as such, allowed rapid growth of the ciliate predator. This trade-off between adapting to a P-limited environment and susceptibility to protist grazing suggests the more efficient removal of low-P prey that potentially has important implications for the functioning of the marine microbial food web in terms of trophic energy transfer and nutrient export efficiency.

A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Manganese Is Essential for PlcP Metallophosphoesterase Activity Involved in Lipid Remodeling in Abundant Marine Heterotrophic Bacteria.

In vast areas of the ocean, microbes must adapt to the availability of scarce nutrients, and a key strategy for reducing the cellular phosphorus (P) quota is to remodel membranes by replacing phospholipids with non-P surrogate lipids. A metallophosphoesterase, PlcP, is essential for lipid remodeling in cosmopolitan marine bacteria of the Roseobacter (e.g., Phaeobacter sp. strain MED193) and SAR11 (e.g., Pelagibacter sp. strain HTCC7211) clades, and transcription of plcP is known to be induced by P limitation. In order to better understand PlcP-mediated lipid remodeling, we sought to characterize PlcP for its metal ion requirement and to determine its selectivity for native bacterial phospholipids. Here, we report the occurrence of a highly conserved binuclear ion center in PlcPs from MED193 and HTCC7211 and show that manganese is the preferred metal for metallophosphoesterase activity. PlcP displayed high activity towards the major bacterial phospholipids, e.g., phosphatidylglycerol but also phosphatidic acid, a key intermediate in phospholipid biosynthesis. In contrast, phosphatidylserine and phosphatidylinositol, both of which are rare lipids in bacteria, are not preferred substrates. These data suggest that PlcP undertakes a generic lipid remodeling role during the cellular response of marine bacteria to P deficiency and that manganese availability may play a key role in regulating the lipid remodeling process.IMPORTANCE Membrane lipids form the structural basis of all cells. In the marine environment, it is well established that phosphorus availability significantly affects lipid composition in cosmopolitan marine bacteria, whereby non-phosphorus-containing lipids are used to replace phospholipids in response to phosphorus stress. Central to this lipid remodeling pathway is a newly identified phospholipase C-type metallophosphoesterase (PlcP). However, little is known about how PlcP activity is regulated. Here, we determined the role of metal ions in regulating PlcP activity and compared PlcP substrate specificities in PlcP enzymes from two model marine bacteria from the marine Roseobacter clade and the SAR11 clade. Our data provide new insights into the regulation of lipid remodeling in these marine bacteria.

[Synthesis and protective effect of ligustrazine intermediates against CoCl2-induced neurotoxicity in differentiated PC12 cell].

Ligustrazine, one of the major effective components of the Chinese traditional medicinal herb Ligusticum Chuanxiong Hort, has been reported plenty of biological activities, such as protect cardiovascular and cerebrovascular, neuroprotection and anti-tumor, et al. Because of its remarkable effects, studies on structural modification of ligustrazine have attracted much attention. Ligustrazine synthetic derivatives reported in recent decades are mainly derived from four primary intermediates (TMP-COOH, TMP-OH, TMP-NH2, HO-TMP-OH). To explore the neuroprotection activitiy of ligustrazine intermediates, six ligustrazine intermediates (2, 5, 8, 11, 12, 13) were synthesized and their protective effects against CoCl2-induced neurotoxicity in differentiated PC12 cells were studied. The target compounds were prepared via different chemical methods, including oxidation, substitution, esterification and amidation without changing the structure nucleus of ligustrazine. Compared with TMP (EC50 = 56.03 micromol x L(-1)), four compounds (2, 5, 12 and 13) exhibited higher activity (EC50 < 50 micromol x L(-1)) respectively, of which, compound 2 displayed the highest protective effect against the damaged PC12 cells (EC50 = 32.86 micromol x L(-1)), but target compounds 8 and 11 appeared lower activity (EC50 > 70 micromol x L(-1)). By structure-activity relationships analysis, the introduction of carboxyl, amino to the side chain of ligustrazine and appropriately increase the proportion of ligustrazine may contribute to enhance its neuroprotective activity, which provides a reference for the design, synthesis and activity screening of relevant series of ligustrazine derivatives in the future.

[Identification of metabolites of antitumor lead compound T-OA in rat urine by HPLC-HRMS].

OBJECTIVE: To study the major metabolites of antitumor lead compound T-OA (oleanolic acyl-3, 5, 6-trimethyl pyrazine-2-methyl ester) in rat urine, in order to preliminarily infer its metabolic mode in rats. METHOD: Rat urines of the blank group, the raw material group (ligustrazine TMP and oleanolic acid OA Moore equivalent) and the T-OA group were collected and freeze-dried; Solids were extracted by ethyl acetate; And then the extracts were re-dissolved with acetonitrile. HPLC-HRMS coupling technique was adopted to find the potential mass spectrum peak under ESI(+) (see symbol) ESI(-) modes. Metabolite-related information was obtained by comparing the three groups of spectra. RESULT: One metabolite of OA and two metabolites of TMP were identified in the raw material group; none metabolite of T-OA but one phase II metabolite was detected in the T-OA group. CONCLUSION: It is the first time to identify one phase II metabolite of T-OA and one phase II metabolite of OA were identified in rat urine. On that basis, the researchers preliminarily inferred that T-OA does not show the efficacy in the form of raw material. The HPLC-HRMS method established could be used to identify metabolites of related derivative structures. This paper could also provide certain reference for designing pro-drugs based on oleanolic acid.

Tenderization effect of cold-adapted collagenolytic protease MCP-01 on beef meat at low temperature and its mechanism.

The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. This study provides insight into the tenderization effect and the mechanism of a cold-adapted collagenolytic enzyme MCP-01 on beef meat at low temperatures. MCP-01 (10 U of caseinolytic activity) reduced the meat shear force by 23% and increased the relative myofibrillar fragmentation index of the meat by 91.7% at 4 °C, and it also kept the fresh colour and moisture of the meat. Compared to the commercially used tenderizers papain and bromelain, MCP-01 showed a unique tenderization mechanism. MCP-01 had a strong selectivity for degrading collagen at 4 °C, showed a distinct digestion pattern on the myofibrillar proteins, and had a different disruption pattern on the muscle fibres under scanning electron micrograph. These results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures.

Study on modified shengmai yin injection for prevention and treatment of brain impairment in endotoxin shock rats.

OBJECTIVE: To examine the effects of modified Shenmai Yin on invigorating vital energy, promoting blood flow, and protection against neural impairment in an endotoxin-induced shock rat model. METHODS: Ninety-six SD rats were randomly divided into four groups: sham operation (saline 20 ml/kg), shock model (lipopolysaccharide, LPS, 8 mg/kg), Reformed Shengmai Yin (Pulse-activating Decoction) (LPS 8 mg/kg + reformed Shengmai Yin Injection 10 ml/kg), and dexamethasone (LPS 8 mg/kg + dexamethasone 5 mg/kg) groups. Each group was subdivided into 1 h, 2 h, 3 h, and 6 h time points for observation. The carotid artery was separated and connected with a biological functional system to monitor mean arterial pressure (MAP). Brain water levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity were also determined. RESULTS: In the shock model group, MAP was progressively decreased after injection of LPS, brain water and MDA contents were increased, brain SOD activity was decreased, and capillary vessel edema in brain tissue was also observed. All these parameters were improved significantly in both treatment groups, although the effects were more marked with Shengmai Yin than with dexamethasone. CONCLUSION: Modified Shengmai Yin exhibits strong anti-shock and neuroprotective effects against Endotoxin-induced shock.

[Nephrotoxicity study of Aristolochia fangchi in rats by metabonomics].

OBJECTIVE: To study the changes of metabolites in rat urine after treatment of Aristolochia fangchi decoction by metabonomic method. METHODS: Sixty-four male SD rats were divided into Aristolochia fangchi group and normal control group. Rats in the Aristolochia fangchi group were orally administered with 8.1 g/(kg.d) of Aristolochia fangchi and the normal control group was administered with equal volume of distilled water for 4 weeks. Twenty-four hour urine was collected at different time points (before, after 2- and 4-week administration and 2 weeks after administration) and their H nuclear magnetic resonance (NMR) spectra were acquired and subjected to data process, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) or orthogonal signal correction (OSC). The contents of blood urea nitrogen (BUN) and serum creatinine (SCr) and histopathological changes of the renal tissues were also detected. RESULTS: The content of BUN of the Aristolochia fangchi group was markedly higher than that of the normal control group after 2-week administration (P<0.05), and cellular edema in tubular endothelial cells, structure damage of glomeruli and inflammatory cell infiltration were found in the kidney. Along with the lasting of administration to 4-week, the renal injury in the Aristolochia fangchi group became more serious, and the contents of BUN and SCr were all significantly higher as compared with the normal control group (P<0.05). Two weeks after administration, the content of BUN in the Aristolochia fangchi group was still higher than that in the normal control group (P<0.05), and the pathological changes in renal tissues were not different from those on the 4th week. The urine of the Aristolochia fangchi group could be readily discriminated from the normal control group at every time point based on PCA. During the whole administration period, compared with the normal control group, the concentration of urinary taurine was increased time-dependently, while the citrate was decreased in the Aristolochia fangchi group. The concentration of hippurate was increased at the 2nd week and the 6th week (2 weeks after drug withdrawal) but decreased at the 4th week; the concentration of creatinine/creatine was increased at the 4th week but decreased at the 6th week; the concentration of 2-oxo-glutarate was decreased and the concentration of trimethylamine oxide was increased at the 4th and 6th week. CONCLUSION: High-dose Aristolochia fangchi can induce renal lesion and its seriousness is correspondent to the lasting of administration. Aristolochia fangchi may also have toxicity on liver.

A novel approach to estimate in vitro antibacterial potency of Chinese medicine using a concentration-killing curve method.

The antibacterial pharmacodynamics against E. coli of Chinese medicine (CM) Rhizoma coptidis (Coptis Root) and its formula Sanhuang, and the control antibiotics enoxacin, were analyzed by a concentration-killing curve (CKC) approach, and the novel parameters BC50 and r for antibacterial potency were proposed. Using the agar plate method, about 400 cells of E. coli were evenly inoculated into LB agar plates containing a series of different concentrations of CM or antibiotic, and after a 24 hour incubation at 37 degrees C, all the viable colonies were enumerated. This resulted in a sigmoid concentration-killing curve , in which No, that could be closely fitted (R2 > 0.9) with the function: N = 1 + e(r(x-BC50))/N0 in which N0, BC50 and r represent meaningfully inoculums size, median bactericidal concentration, and bactericidal intensity, respectively. N modeled the survival of colony-forming units on each plate (CFU/plate) in a concentration series x of the drug. The CKC was symmetrical about its single inflexion (BC50, N0/2). Therefore theoretically, 2BC50 can replace MBC (minimum bactericidal concentration). BC1 = BC50 + r/ln(N0-1), the drug concentration at r which only one colony survived, was the least critical value of MBC in CKC. The parameters 2BC50 and BC1 agreed more closely with the definition of MBC, and were little affected by either the biochemical basis of the antibacterial or the inoculum's size (200-400 CFU/plate), and were determined by a multi-point curve. As a result, these were more accurate, reproducible and practical as metrics than was the endpoint of MBC. The two-dimensional CKC, involving BC50 and r, captures the intrinsic dynamics of the antibacterial effect of CM/strain versus concentration, and it is consistent with the Logistic equation of the bacterial growth curve in the format. This verified approach has considerable value as a tool for the accurate and proper administration of CM. The CKC of CM, different from that of antibiotics, is likely to be the resultant force of each ingredient in certain CM, which provides a clue to solve the problem of antibiotic resistance.