13C-NMR studies on the influence of pH and nitrogen source on polyol pool formation in Aspergillus nidulans

Abstract The composition of the polyol pools in Aspergillus nidulans mycelium during active growth on sucrose depends strongly on pH. At pH 2.5, only mannitol is present. A comparison between nitrate- and ammonium-grown cultures shows stimulation of the arabitol content with nitrate a former nitrogen source. When starved mycelium is incubated either with natural-abundance or ¹³C-enriched glucose, label appears rapidly in mannitol and arabitol, regardless of the nitrogen source or the pH used.     

Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

Synthesis and characterization of biodegradable and thermosensitive polymeric micelles with covalently bound doxorubicin-glucuronide prodrug via click chemistry

Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human β-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of β-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of β-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.     

Immunoblockade of PSGL-1 attenuates established experimental murine colitis by reduction of leukocyte rolling

Recruitment of circulating leukocytes into the colonic tissue is a key feature of intestinal inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) and very late antigen-4 (VLA-4) are expressed on leukocytes and play an important role in leukocyte-endothelial cell adhesive interactions. We examined the effects of immunoneutralization of PSGL-1 and VLA-4 on leukocyte recruitment in vivo in the development and treatment of experimental colitis. Chronic colitis was induced in balb/c mice by oral administration of dextran sodium sulfate (DSS). Monoclonal antibodies 2PH1 (anti-PSGL-1) and PS/2 (anti-VLA-4) or the combination of both were injected intravenously, and leukocyte adhesion was observed for 60 min in colonic submucosal venules by intravital microscopy (IVM) under isoflurane/N(2)O anesthesia. In addition, mice with established colitis were treated by daily intraperitoneal injections of 2PH1, PS/2, or the combination of both over 5 days. Disease activity index (DAI), histology, and myeloperoxidase (MPO) levels were compared with sham-treated DSS controls. We found that 2PH1 reduced the number of rolling leukocytes (148.7 +/- 29.8 vs. 36.9 +/- 8.7/0.01 mm(2)/30 s, P < 0.05), whereas leukocyte velocity was increased (24.0 +/- 3.6 vs. 127.8 +/- 11.7 microm/s, P < 0.05). PS/2 reduced leukocyte rolling to a lesser extent. Leukocyte firm adhesion was not influenced by 2PH1 but was strongly reduced by PS/2 (24.1 +/- 2 vs. 4.4 +/- 0.9/0.01 mm(2)/30 s, P < 0.05). Combined application did not cause additional effects on leukocyte adhesion. Treatment of chronic colitis with 2PH1 or PS/2 reduced DAI, mucosal injury, and MPO levels significantly. Combined treatment led to a significantly better reduction of DAI (0.4 +/- 0.1 vs. 2.1 +/- 0.2 points) and histology (9.7 +/- 0.9 vs. 21.4 +/- 4.6 points). In conclusion, PSGL-1 and VLA-4 play an important role for leukocyte recruitment during intestinal inflammation. Therapeutic strategies designed to disrupt interactions mediated by PSGL-1 and/or VLA-4 may prove beneficial in treatment of chronic colitis.     

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.     

PECAM-1 (CD 31) mediates transendothelial leukocyte migration in experimental colitis

Transendothelial migration of circulating leukocytes into the colonic wall is a key step in the development of the inflammatory infiltrate in inflammatory bowel disease (IBD). The platelet-endothelial cell adhesion molecule-1 PECAM-1 (CD31) is expressed in the tight junction area of endothelial cells, where it is supposed to support the transmigration process. The aim of this study was to determine the role of PECAM-1 in experimental IBD and to show whether blockade of PECAM-1 has therapeutic effects. Chronic colitis was induced in female BALB/c mice by cyclic oral administration of dextran sodium sulfate (DSS) 3% (wt/vol). Expression of PECAM-1 was visualized by immunohistochemistry. In the treatment group animals received 1 mg/kg anti-PECAM-1 (2H8) ip daily starting on day 26. On day 30 leukocyte adhesion and migration was measured during N(2)O-isoflurane anesthesia in the distal colon by intravital microscopy. Disease activity index (DAI), histology, and MPO levels were compared with healthy and diseased controls. PECAM-1 was expressed in colitic mice. Chronic DSS colitis was characterized by a marked increase in rolling, adherent, and transmigrated leukocytes compared with healthy controls. Immunoblockade of PECAM-1 reduced leukocyte transmigration significantly and also diminished leukocyte rolling and sticking in an indirect manner. It also resulted in a significantly diminished DAI and MPO levels, as well as an amelioration of the histological inflammation score. PECAM-1 plays an important role in transendothelial leukocyte migration in DSS colitis. PECAM-1 could be a novel target for antibody-based treatment in IBD.     

Mechanisms underlying the anti-inflammatory actions of boswellic acid derivatives in experimental colitis

Recent clinical trials of the gum resin of Boswellia serrata have shown promising results in patients with ulcerative colitis. The objective of this study was to determine whether a semisynthetic form of acetyl-11-keto-beta-boswellic acid (sAKBA), the most potent anti-inflammatory component of the resin, also confers protection in experimental murine colitis induced by dextran sodium sulfate (DSS) to compare its effects with those standard medications of ulcerative colitis like steroids and to examine whether leukocyte-endothelial cell adhesion is a major target of action of sAKBA. Clinical measurements of disease activity and histology were used to assess disease progression, and intravital microscopy was employed to monitor the adhesion of leukocytes and platelets in postcapillary venules of the inflamed colon. sAKBA treatment significantly blunted disease activity as assessed both grossly and by histology. Similarly, the recruitment of adherent leukocytes and platelets into inflamed colonic venules was profoundly reduced in mice treated with sAKBA. Because previous studies in the DSS model have shown that P-selectin mediates these blood cell-endothelial cell interactions, the expression of P-selectin in the colonic microcirculation was monitored using the dual-radiolabeled antibody technique. The treatment of established colitis with sAKBA largely prevented the P-selectin upregulation normally associated with DSS colitis. All of the protective responses observed with sAKBA were comparable to that realized in mice treated with a corticosteroid. Our findings demonstrated an anti-inflammatory effect of sAKBA and indicated that P-selectin-mediated recruitment of inflammatory cells is a major site of action for this novel anti-inflammatory agent.     

Adaptation of cardiac structure by mechanical feedback in the environment of the cell: a model study.

In the cardiac left ventricle during systole mechanical load of the myocardial fibers is distributed uniformly. A mechanism is proposed by which control of mechanical load is distributed over many individual control units acting in the environment of the cell. The mechanics of the equatorial region of the left ventricle was modeled by a thick-walled cylinder composed of 6-1500 shells of myocardial fiber material. In each shell a separate control unit was simulated. The direction of the cells was varied so that systolic fiber shortening approached a given optimum of 15%. End-diastolic sarcomere length was maintained at 2.1 microns. Regional early-systolic stretch and global contractility stimulated growth of cellular mass. If systolic shortening was more than normal the passive extracellular matrix stretched. The design of the load-controlling mechanism was derived from biological experiments showing that cellular processes are sensitive to mechanical deformation. After simulating a few hundred adaptation cycles, the macroscopic anatomical arrangement of helical pathways of the myocardial fibers formed automatically. If pump load of the ventricle was changed, wall thickness and cavity volume adapted physiologically. We propose that the cardiac anatomy may be defined and maintained by a multitude of control units for mechanical load, each acting in the cellular environment. Interestingly, feedback through fiber stress is not a compelling condition for such control.     

Novel fast degradable thermosensitive polymeric micelles based on PEG-block-poly(N-(2-hydroxyethyl)methacrylamide-oligolactates)

The aim of this study was to design a thermosensitive polymeric micelle system with a relatively fast degradation time of around 1 day. These micelles are of interest for the (targeted) delivery of biologically active molecules. Therefore, N-(2-hydroxyethyl)methacrylamide-oligolactates (HEMAm-Lac(n)()) were synthesized and used as building blocks for biodegradable (block co) polymers. p(HEMAm-Lac(2)) is a thermosensitive polymer with a cloud point (CP) of 22 degrees C which could be lowered by copolymerization with HEMAm-Lac(4). The block copolymer PEG-b-((80%HEMAm-Lac(2))-(20%HEMAm-Lac(4))) self-assembled into compact spherical micelles with an average size of 80 nm above the CP of the thermosensitive block (6 degrees C). Under physiological conditions (pH 7.4; 37 degrees C), the micelles started to swell after 4 h and were fully destabilized within 8 h due to hydrolysis of the lactate side chains. Rapidly degrading thermosensitive polymeric micelles based on PEG-b-((80%HEMAm-Lac(2))-(20%HEMAm-Lac(4))) have attractive features as a (targeted) drug carrier system for therapeutic applications.