Genetic association study of adaptor protein complex 4 with cerebral palsy in a Han Chinese population

Adaptor protein complex 4 (AP-4) plays a key role in vesicle formation, trafficking, and sorting processes that are critical for brain development and function. AP-4 consists of four subunits encoded by the AP4E1, AP4B1, AP4M1, and AP4S1 genes. A number of studies have pointed to the involvement of AP-4–mediated vesicular trafficking pathways in the etiology of cerebral palsy (CP), the most notable of which are the causative mutations that have recently been identified in each of the AP-4 genes in different CP families. We postulated, therefore, that variations in AP-4 genes might influence an indivual’s susceptibility to CP. In the present study, 16 SNPs were genotyped among 517 CP patients and 502 healthy controls from the Han Chinese population. We systematically analyzed the association of the AP4E1, AP4B1, AP4M1, and AP4S1 genes with CP on the basis of clinical characteristics. No significant associations were found between these variants and the overall risk of CP. Subgroup analysis showed that rs1217401 of AP4B1 was significantly associated with CP as a sequela of hypoxic-ischemic encephalopathy (HIE) (CP + HIE) (allele: p = 0.042151; genotype: p = 4.46 × 10^?6). Our results indicate that the 16 variants studied in the genes of the four subunits of AP-4 have no detectable effects on the overall susceptibility to CP, but AP4B1 appears to be a susceptibility gene for CP + HIE in the Han Chinese population.     

Bacteriorhodopsin wildtype and variant aspartate-96 → aspargine as reversible holographic media

Air dried films of purple membranes (PM) from Halobacterium halobium containing the photochromic protein bacteriorhodopsin (BR) were prepared and the BR-photocycle of this material analyzed. The absorption maxima of the initial state B (λ_max = 570 nm) and the photochemical intermediate M (λ_max = 412 nm), which is the longest living intermediate in suspension (τ ≈ 10 ms), were spectrally well separated. Light-induced population gratings between B and M were used for reversible holographic recording in these dry PM films. The resolution (>5,000 lines/mm) of PM films was comparable to the corresponding values of conventional photochromic recording materials. The longterm stability toward photochemical degradation of PM films is excellent (> 100.000 recording cycles). The spectral bandwidth (400-680 nm) of such films covers nearly the whole visible spectrum. Both the photochemical transition from B → M with wavelengths in the green-red range and from M → B with blue light were utilized for holographic recording. The latter possibility (M → B) seems to be advantageous for several applications because the holographic grating is only formed during reconstruction. Higher reading intensities lead to higher population of the M-state and result in an increase of the fringe contrast instead of decreasing it. New possibilities for the further development of holographic media based on bacteriorhodopsin are raised by the availability of PM variants with modified optical properties. By the use of the variant BR-326, which differs from the wildtype PM by a single amino acid exchange (aspartate-96 → asparagine), the sensitivity of PM films is increased by ~50% from 12 cm²/J to 19 cm²/J for recording with 568 nm. The sensitivity for recording with 413 nm (33 cm²/J) is not influenced by the amino acid exchange. The observed diffraction efficiency η of PM films with BR-326 is twice that of BR-wildtype (BR-WT) films and is in the range of conventional organic photochromics (≈ 1%). In dried films of both BR-WT and BR-326 the M-decay was shown to be at least biexponential.     

The blood metabolome of incident kidney cancer: A case–control study nested within the MetKid consortium

BackgroundExcess bodyweight and related metabolic perturbations have been implicated in kidney cancer aetiology, but the specific molecular mechanisms underlying these relationships are poorly understood. In this study, we sought to identify circulating metabolites that predispose kidney cancer and to evaluate the extent to which they are influenced by body mass index (BMI).Methods and findingsWe assessed the association between circulating levels of 1,416 metabolites and incident kidney cancer using pre-diagnostic blood samples from up to 1,305 kidney cancer case–control pairs from 5 prospective cohort studies. Cases were diagnosed on average 8 years after blood collection. We found 25 metabolites robustly associated with kidney cancer risk. In particular, 14 glycerophospholipids (GPLs) were inversely associated with risk, including 8 phosphatidylcholines (PCs) and 2 plasmalogens. The PC with the strongest association was PC ae C34:3 with an odds ratio (OR) for 1 standard deviation (SD) increment of 0.75 (95% confidence interval [CI]: 0.68 to 0.83, p = 2.6 × 10⁻⁸). In contrast, 4 amino acids, including glutamate (OR for 1 SD = 1.39, 95% CI: 1.20 to 1.60, p = 1.6 × 10⁻⁵), were positively associated with risk. Adjusting for BMI partly attenuated the risk association for some—but not all—metabolites, whereas other known risk factors of kidney cancer, such as smoking and alcohol consumption, had minimal impact on the observed associations. A mendelian randomisation (MR) analysis of the influence of BMI on the blood metabolome highlighted that some metabolites associated with kidney cancer risk are influenced by BMI. Specifically, elevated BMI appeared to decrease levels of several GPLs that were also found inversely associated with kidney cancer risk (e.g., −0.17 SD change [ß_BMI] in 1-(1-enyl-palmitoyl)-2-linoleoyl-GPC (P-16:0/18:2) levels per SD change in BMI, p = 3.4 × 10⁻⁵). BMI was also associated with increased levels of glutamate (ß_BMI: 0.12, p = 1.5 × 10⁻³). While our results were robust across the participating studies, they were limited to study participants of European descent, and it will, therefore, be important to evaluate if our findings can be generalised to populations with different genetic backgrounds.ConclusionsThis study suggests a potentially important role of the blood metabolome in kidney cancer aetiology by highlighting a wide range of metabolites associated with the risk of developing kidney cancer and the extent to which changes in levels of these metabolites are driven by BMI—the principal modifiable risk factor of kidney cancer.

In a case-control study, Florence Guida and colleagues identify metabolites associated with risk of kidney cancer, and use Mendelian randomization techniques to study the role of body mass index in this relationship.     

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease,ARP is involved in the plant nucleotide incision repair pathway

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3' → 5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (k_cat/K_M = 134 and 7.3 μM⁻¹·min⁻¹, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3' → 5' exonuclease activities (k_cat/K_M = 314 and 34 μM⁻¹·min⁻¹, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp^–/– mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H₂O₂, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.     

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na⁺-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [³H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na⁺-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [³H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na⁺-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.     

The dynamics of carbohydrate metabolism in Candida albicans

The total cellular concentrations of the intermediary metabolites and the carbohydrate end products were determined for starved Candida albicans yeast cells and cells forming germ tubes during a 60-min incubation in imidazole-HCl buffer in the absence and presence of 2.5 mM glucose, 2.5 mM glutamine, and 0.2% serum at 37°C. These cells were also incubated in the presence of tracer [U-¹⁴C]glucose and the specific radioactivities of the metabolites and end products determined. The labeling data indicated (1) a minimum of two metabolically independent pools of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and uridine diphosphoglucose; (2) compartmentation of the pathways of catabolism and anabolism; (3) channeling of the exogenous tracer glucose into the anabolic pathway compartments of the starved cells; and (4) a significant rate of turnover of cell wall carbohydrates in cells incubated under nongrowth conditions and rapid turnover of these pools in germ tube forming cells. The labeling data will be used to construct kinetic models of carbohydrate metabolism in C. albicans.     

CONTRIBUTION OF CIRCULATING LEUKOCYTES TO CYTOKINE PRODUCTION IN PANCREATIC DUCT OBSTRUCTION-INDUCED ACUTE PANCREATITIS IN RATS

Little information is available regarding the role of circulating leukocytes in the pathogenesis of acute pancreatitis (AP). Our aim was to explore the time-course of the potential role of inflammatory peripheral blood (PB) cells during AP induced in rats by pancreatic duct obstruction (PDO). Flow cytometry immunophenotyping was used to analyse the distribution of the major circulating leukocyte subsets, the activation state of circulating monocytes as reflected by both CD11b expression and TNF-α production and the relative contribution of T-cell derived pro- (TNF-α) and anti- (IL-10) inflammatory mediators at different stages of PDO-induced AP. A progressive increase in PB neutrophils and monocytes was observed up to 6 h after PDO whereas lymphocytes, as well as CD4⁺ and CD8⁺ T-cell subsets, rose as early as 1.5 h after PDO and decreased thereafter. Monocytes were activated in PB from 6 h after inducing AP as reflected by increases in both CD11b expression and spontaneous TNF-α production; nevertheless, they showed the capability of producing TNF-α at earlier AP stages by lipopolysaccharide (LPS) stimulation. In contrast, T-cells were unable to produce TNF-α during AP neither spontaneously nor after stimulation with PMA/Ionomycin. Therefore, only PB monocytes contribute to increase TNF-α levels in plasma as observed from 12 h onwards after inducing AP. Interleukin-10 was produced by T-cells 6 h after PDO only after PMA/Ionomycin stimulation. We conclude that systemic inflammatory events are triggered off at early stages of PDO-induced AP, with the activation of circulating monocytes, though not T-cells, playing a central role.     

Changes in dormancy of Sisymbrium officinaleseeds do not depend on changes in respiratory activity

Effects of dark incubation at different temperatures were studied on dormancy and respiratory activity of seeds of Sisymbrium officinale (L.) Scop. Because germination of this species absolutely depends on the simultaneous action of light and nitrate, changes in dormancy could be studied in darkness without the interference of early germination events. Upon the start of incubation rates of O₂ uptake and CO₂ release rose. This was followed by a gradual decrease until stable levels of O₂ uptake and CO₂ release were achieved. Seeds kept for prolonged periods at 24^°C, showed neither a change in germination capacity nor in rates of O₂ uptake and CO₂ release. Respiratory quotients were 0.55–0.7. The initial rise in O₂ uptake correlated with the rate of water uptake and with breaking of primary dormancy. However, the subsequent decline in O₂ uptake was not generally linked to induction of secondary dormancy. An increased O₂ uptake was not required during breaking of secondary dormancy. It is concluded that changes in dormancy are not generally related to changes in respiratory activity. However, germination strongly depends on respiration. The increase in O₂ uptake started well before radicle protrusion. A far red irradiation only reversed this increase when it was given before germination escaped from its red light antagonising action. The contribution of different respiratory pathways was followed during prolonged incubation at 24^°C in darkness. KCN at 1.5 mM was needed to inhibit the cytochrome pathway (CP) and benzohydroxamic acid (BHAM) at 30 mM to inhibit the alternative pathway (AP). These concentrations did not exert any side effects. Electron flow was predominantly via the CP, maximally 10% was via the AP. Flow through the CP declined during the first 6 days and residual respiration remained constant. Therefore, the contribution of residual respiration became relatively more important with prolonged incubation. KCN at concentrations that almost completely inhibited flow through the CP, did not dramatically reduce germination. BHAM already inhibited germination at concentrations that do not inhibit oxygen uptake.     

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.     

Binding of antitumor tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen to human serum albumin

Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. The aim of this study was to examine the interaction of human serum albumin (HSA) with tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen at physiological conditions, using constant protein concentration and various drug contents. FTIR, UV-Visible, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on HSA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bound HSA via both hydrophobic and hydrophilic interactions with overall binding constants of K_tam = 1.8 (±0.2) × 10⁴ M⁻¹, K_4-hydroxytam = 1.8 (±0.4) × 10⁴ M⁻¹ and K_endox = 2.0 (±0.5) × 10⁴ M⁻¹. The number of bound drugs per protein is 1.2 (tamoxifen), 1.7 (4-hydroxitamoxifen) and 1.0 (endoxifen). Structural modeling showed the participation of several amino acid residues in drug-HSA complexation, with extended H-bonding network. HSA conformation was altered by tamoxifen and its metabolites with a major reduction of α-helix and an increase in β-sheet, random coil and turn structures, indicating a partial protein unfolding. Our results suggest that serum albumins can act as carrier proteins for tamoxifen and its metabolites in delivering them to target tissues.     

Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

Summary The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1^–1.h^–1 for several weeks. Correspondence to: H.-J. Rehm     

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10⁻⁶M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.     

EDTA improves stability of whole blood C-Peptide and insulin to over 24 hours at room temperature

Introduction

C-peptide and insulin measurements in blood provide useful information regarding endogenous insulin secretion. Conflicting evidence on sample stability and handling procedures continue to limit the widespread clinical use of these tests. We assessed the factors that altered the stability of insulin and C-peptide in blood.

Methods

We investigated the impact of preservative type, time to centrifugation, storage conditions and duration of storage on the stability of C-peptide and insulin on three different analytical platforms.

Results

C-peptide was stable for at least 24 hours at room temperature in both centrifuged and whole blood collected in K⁺-EDTA and serum gel tubes, with the exception of whole blood serum gel, which decreased to 78% of baseline at 24 hours, (p = 0.008). Insulin was stable at room temperature for 24 hours in both centrifuged and whole blood collected in K⁺-EDTA tubes. In contrast insulin levels decreased in serum gel tubes both centrifuged and whole blood (66% of baseline, p = 0.01 and 76% of baseline p = 0.01, by 24 hours respectively). C-peptide and insulin remained stable after 6 freeze-thaw cycles.

Conclusions

The stability of C-peptide and insulin in whole blood K⁺-EDTA tubes negates the need to conform to strict sample handling procedures for these assays, greatly increasing their clinical utility.     

Ca2+ signalling underlying pancreatitis

In spite of significant scientific progress in recent years, acute pancreatitis (AP) is still a dangerous and in up to 5% of cases deadly disease with no specific cure. It is self-resolved in the majority of cases, but could result in chronic pancreatitis (CP) and increased risk of pancreatic cancer (PC). One of the early events in AP is premature activation of digestive pro-enzymes, including trypsinogen, inside pancreatic acinar cells (PACs) due to an excessive rise in the cytosolic Ca²⁺ concentration, which is the result of Ca²⁺ release from internal stores followed by Ca²⁺ entry through the store operated Ca²⁺ channels in the plasma membrane. The leading causes of AP are high alcohol intake and biliary disease with gallstones obstruction leading to bile reflux into the pancreatic duct. Recently attention in this area of research turned to another cause of AP – Asparaginase based drugs – which have been used quite successfully in treatments of childhood acute lymphoblastic leukaemia (ALL). Unfortunately, Asparaginase is implicated in triggering AP in 5–10% of cases as a side effect of the anti-cancer therapy. The main features of Asparaginase-elicited AP (AAP) were found to be remarkably similar to AP induced by alcohol metabolites and bile acids. Several potential therapeutic avenues in counteracting AAP have been suggested and could also be useful for dealing with AP induced by other causes. Another interesting development in this field includes recent research related to pancreatic stellate cells (PSCs) that are much less studied in their natural environment but nevertheless critically involved in AP, CP and PC. This review will attempt to evaluate developments, approaches and potential therapies for AP and discuss links to other relevant diseases.     

Activities of respiratory pathways and the pool of nonstructural carbohydrates in greening leaves of spring wheat seedlings

Seedlings of spring wheat (Triticum aestivum L.) were used to study the dynamics of leaf respiration, the respiratory pathway ratio, and relation of activities of these pathways to the content of soluble carbohydrates in the leaf during greening of seedlings for 48 h under continuous photosynthetically active light (190 μmol/(m² s)). Changes in leaf respiration during de-etiolation were closely related to modulation of the alternative respiratory pathway (AP) activity. The rate of cytochrome respiratory pathway (CP) depended directly on the carbohydrate content and growth rate. These relations suggest that the substrate regulation of CP activity during greening is mediated by the energy needs for growth and is effectively regulated by the mechanism of respiratory control. The highest rates of AP were observed after a 6-h exposure of seedlings to light. The proportion of CP/AP at this stage was close to unity. The temporal pattern of AP activity during de-etiolation was independent on the content of soluble carbohydrates. Hence, in addition to substrate regulation of AP, there are other intricate mechanisms of AP involvement. Our results are in accordance to the state that the alternative respiratory pathway participates in maintaining homeostasis in phototrophic cells during development of the photosynthetic function.     

Single-Phase and Two-Phase Anaerobic Co-Digestion of Residues from the Treatment Process of Waste Vegetable Oil and Pig Manure

The co-digestion of residues from the pre-treatment process of waste vegetable oil (OW) and pig manure (PM) was performed under different OW/PM feed ratios (1:0, 1:1 and 1:3 v/v) and at organic loading rates ranging from 0.25 to 3.1 kg VS m^?3 day^?1 in lab-scale single-phase (SP) and two-phase (TP) systems. From the experiments, it was observed that digestion of OW alone was inhibitory for the anaerobic degradation. Mixing OW with PM neutralized the negative effects of lipids accumulation and high VS removal efficiencies were realized in both systems (63 and 71 % in SP system and 69 and 72 % in TP system, at 1:1 and 1:3 OW/PM mixtures, respectively). Under the same operational conditions, the methane yield was 0.30 and 0.22 m³ CH₄ kg^?1 VS removed for the SP anaerobic digester and 0.30 and 0.27 m³ CH₄ kg^?1 VS removed for the TP configuration. Additionally, TP digestion presented more stable operation and higher treatment capacity.     

Pteris multifida,Cortex phellodendri,and probiotics attenuated inflammatory status and immunity in mice with a Salmonella enterica serovar Typhimurium infection

Pteris multifida (PM) and Cortex phellodendri (CP) are medicinal foods used for gastrointestinal protection. Lactic-acid bacteria are probiotics. Salmonella Typhimurium strain ST21-infected mice were used to examine the alleviative effects of two lactic-acid bacteria (LAB) as well as aqueous extracts of PM and CP for a 4-day treatment. CP and LAB decreased fecal ST counts. CP and PM reduced the ST21 count in the blood, intestine, and liver. LAB lowered the ST21 count in the intestine and spleen. CP and LAB decreased the IFN-gamma level; PM lowered the TNF-alpha level; and both LAB and PM reduced the IL-1beta level in serum. PM and CP lowered the IgG level in serum. The data in a macrophage infection model indicate that TNF-alpha was partial involved in this alleviative effects, other mechanisms might be involved. In sum, these novel findings suggest that PM, CP, and LAB probiotics are potential anti-Salmonellae agents.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.