Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s with functional carbonate building blocks. 1. Chemical synthesis and their structural and physical characterization

This study presents chemical synthesis, structural, and physical characterization of novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s P(BS-co-CC) bearing functional carbonate building blocks. First, five kinds of six-membered cyclic carbonate monomers, namely, trimethylene carbonate (TMC), 1-methyl-1,3-trimethylene carbonate (MTMC), 2,2-dimethyl-1,3-trimethylene carbonate (DMTMC), 5-benzyloxytrimethylene carbonate (BTMC), and 5-ethyl-5-benzyloxymethyl trimethylene carbonate (EBTMC), were well prepared from ethyl chloroformate and corresponding diols at 0 degrees C in THF solution with our modified synthetic strategies. Then, a series of new P(BS-co-CC)s were synthesized at 210 degrees C through a simple combination of poly-condensation and ring-opening-polymerization (ROP) of hydroxyl capped PBS macromers and the prepared carbonate monomers, and titanium tetra-isopropoxide Ti(i-OPr)4 was used as a more suitable catalyst of 5 candidate catalysts which could concurrently catalyze poly-condensation and ROP. By means of NMR, GPC, FTIR, and thermal analytical instruments, macromolecular structures and physical properties have been characterized for these aliphatic poly(ester carbonate)s. The experimental results indicated that novel biodegradable P(BS-co-CC)s were successfully synthesized with number average molecular weight Mn ranging from 24.3 to 99.6 KDa and various CC molar contents without any detectable decarboxylation and that the more bulky side group was attached to a cyclic carbonate monomer, the lower reactivity for its copolymerization would be observed. The occurrences of 13C NMR signal splitting of succinyl carbonyl attributed to the BS building blocks could be proposed due to the randomized sequences of BS and CC building blocks. FTIR characterization indicated two distinct absorption bands at 1716 and 1733 approximately 1735 cm(-1), respectively, stemming from carbonyl stretching modes for corresponding BS and CC units. With regard to their thermal properties, it is seen that the synthesized P(BS-co-CC)s exhibited thermal degradation temperatures 10 approximately 20 degrees C higher than that of PBS. On the basis of the synthesized P(BS-co-BTMC)s, new aliphatic poly(butylene succinate-co-5-hydroxy trimethylene carbonate)s were further synthesized, bearing hydrophilic hydroxyl pendant functional groups through an optimized Pd/C catalyzed hydrogenation. These semi-crystalline new biodegradable aliphatic copolymers with tunable physical properties and functional carbonate building blocks might be expected as potential new biomaterials.     

Glucose effect on the expression of 150 kDa oxygen-regulated protein in HeLa cells

Chaperones assist in the correct folding of newly synthesised proteins in the endoplasmic reticulum (ER) of cells, this being essential for the translocation of protein molecules to specific subcellular compartments, extracellular matrix or to biological fluids. The biosynthesis of some ER chaperones is regulated by glucose. They are named "glucose-regulated proteins" (GRPs). The function of some GRPs depends on oxygen, a subgroup named "oxygen-regulated proteins" (ORPs). The biosynthesis of ORPs is induced by deprivation of glucose or oxygen. Exposure of HeLa cells to glucose starvation induces the biosynthesis of various GRPs including ORP 150. The expression of ORP 150 is regulated by the concentration of glucose in the culture medium, being induced by a shortage and repressed by a presence of glucose. We have shown that both glucose starvation and transfection of cells with siRNA (specific to ORP 150 mRNA) evoke similar, but quantitatively different, effects. The cells grown for 72 h in a 4.5 mg/ml glucose-containing medium demonstrated low apoptosis (3.7%) whereas in a 0.5 mg/ml glucose-containing medium the apoptosis was increased to 10%. The effect of transfection on apoptosis was distinctly higher with almost 22% of apoptotic cells detected in 72 h cultures. One may conclude that ORP 150 reduces the pro-apoptotic effects of glucose starvation. Such a hypothesis is supported by the observation that the transfection procedure makes HeLa cells resistant to the regulatory effect of glucose on ORP 150 production. The transfected cells do not respond to glucose starvation with an overexpression of ORP 150. It is apparent from our experiments that ORP 150 plays an important role in adaptation of cells to the shortage of glucose and reduces the pro-apoptotic effect of glucose starvation.     

Diagnostic and therapeutic doubts in retrobulbar neuritis in children

Retrobulbar neuritis is often very complicated clinical entity. The most common cause of retrobulbar neuritis is demyelinating disease of CNS. This report is to express some other uncommon causes of it. Three children, age 8 to 12 with sudden and severe visual loss are presented. The diagnosis of retrobulbar neuritis is made by complete ophtalmological examination in consultation with neuropediatrics and neuroradiologist. Different ethiological causes of retrobulbar neuritis are found: pranasal sinusitis, functional visual loss and pseudotumor cerebri. In first two children complete therapeutically effort was as expected, and by child with pseudotumor cerebri there was no improvement of visual acuity, even after 6 months. In this presentation the authors want to emphasise some uncommon causes of retrobulbar neuritis.     

Acute antenatal hypoxia on different stages of embryogenesis changes the offspring behavior patterns and biogenic amine levels

The effects of acute hypobaric hypoxia on the stages of progestation or beginning of organogenesis on biogenic amines levels in brain stem and cerebral cortex of their mature offspring as well as on their behavior, were investigated. It was shown that acute hypoxia applied in the period of embryonic organogenesis resulted in severe delayed changes in offspring spontaneous behavior and led to marked changes of biogenic amine levels, particularly expressed for dopaminergic system. Females subjected to antenatal hypoxia happened to be more sensitive to its influences than males.     

Cytokine gene polymorphisms as potential risk and protective factors in renal cell carcinoma

Serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 levels in the serum of 34 patients with head and neck cancer (HNC) undergoing locoregional radiotherapy (RT) were examined. The aim of the RT was definitive in 19 and postoperative adjuvant in 15 patients. Serum TNF-alpha and IL-6 levels were recorded before RT and after the completion of the fifth week of RT. The mean TNF-alpha levels before and after RT were 28.26 +/- 2.87 and 83.03 +/- 7.47, and the mean IL-6 levels were 61.56 +/- 14.32 and 122.45 +/- 30.66, respectively. The statistical analysis yielded a significant rise in TNF-alpha levels with RT in all patients (p < 0.0001) and also in IL-6 levels in patients treated with postoperative adjuvant RT (p = 0.001). Irradiation is likely to cause an acute phase response, and the cytokines studied may be used to monitor this clinically important response in further trials.     

Temperature, media, and point of induction affect the N-terminal processing of interleukin-1beta

The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three isoforms when expressed in the Escherichia coli strain BL-21(DE3). These isoforms are produced by the non-homogeneous processing of the N-terminal methionine residue by the endogenous bacterial aminopeptidase and differ in the first residue (1-met, 1-ala, and 1-pro). Importantly, these isoforms have significantly different binding affinities for the IL receptor protein. Varying the temperature, media composition, and point of induction affects this N-terminal processing to favor one of the three isoforms of IL-1beta. We found changes in media composition and/or point of induction affected the abundance of the isoforms by as much as 15-fold. The 1-pro isoform decreased from 60.9 to 4.7% in Luria broth (LB) and minimal media (MM), respectively, when protein expression was induced at an OD600 of 0.9. Conversely, the abundance of the 1-met isoform is much higher in MM than in LB showing the reverse effect (2.6 and 50.7% in LB and MM, respectively, at an OD600 of 0.9), and the degree to which they are favored depends significantly upon the induction point. Our results show that it is possible to favor the expression of various N-terminal isoforms of IL-1beta by adjusting the environmental growth conditions. Given that the initiator methionine residue is necessary for expression in bacterial hosts and is known to affect the stability of other recombinant proteins our approach may be a useful general method for determining the optimal conditions for expressing and purifying pure, homogenous samples of recombinant proteins for structural and biological studies.     

Functional characterization of EhADH112: an Entamoeba histolytica Bro1 domain-containing protein

EhADH112 is part of the EhCPADH complex, a protein involved in key events of the Entamoeba histolytica host invasion. EhADH112 participates in trophozoite adherence to target cells and in phagocytosis. We report here the finding of two EhADH112 homologues in the E. histolytica genome (EhADH112-like proteins). EhADH112 and its relatives have a Bro1 domain at their amino-terminus and a consensus context for phosphorylation by Src-tyrosine kinases, both involved in signal transduction processes in other organisms. Our findings associate EhADH112 to supplementary functions related to those reported for the Alix/AIP1 family. To elucidate the precise function of EhADH112, we studied the phenotypes displayed by trophozoites transfected with the Ehadh112 full gene. Transfected trophozoites overexpressed a 78 kDa protein, which was mainly targeted to the EhCPADH complex. Moreover, these trophozoites exhibited enhanced phagocytic rates, providing further evidence of EhADH112 contribution to adhesion and phagocytosis activities.     

Activities of antioxidant enzymes in two stages of pathology development in sucrose-fed rats

The activities of catalase in liver, heart and kidney as well as glutathione peroxidase and superoxide dismutase in liver, heart, kidney, and serum from hypertriglyceridemic and hypertensive female and male rats were measured at 3 and 8 months of daily administration of sucrose in their drinking water. This treatment induces high levels of serum triglycerides, central obesity, moderate hypertension, hyperinsulinemia, and an increase in lipoperoxidation, among other alterations. The experimental periods were chosen on the basis of previous observations: at 3 months the level of serum triglycerides increases significantly above the normal value and remains without major changes thereafter, but the blood pressure only rises significantly at about 4 months in males and 5 months in females. So, at 8 months the rats have been subjected to abnormal conditions for 3-4 months. The effect of these and the influence of sex on levels of antioxidant enzymes were investigated. Both factors, sucrose treatment and sex, were conducive to significant changes in those variables.     

Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 microm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

The small intestine plays an important role in upregulating CGRP during sepsis

Although studies have indicated that calcitonin gene-related peptide (CGRP), a potent vasodilatory peptide, is upregulated after endotoxic shock, it remains controversial whether this peptide increases during sepsis and, if so, whether the gut is a significant source of CGRP under such conditions. To study this, polymicrobial sepsis was induced by cecal ligation and puncture (CLP) followed by fluid resuscitation. Plasma levels of CGRP were measured at 2, 5, and 10 h after CLP (i.e., early, hyperdynamic sepsis) and at 20 h after CLP (late, hypodynamic sepsis). The results indicate that plasma CGRP did not increase at 2--5 h but increased by 177% at 10 h after CLP (P < 0.05). At 20 h after the onset of sepsis, however, the elevated plasma CGRP returned to the sham level. To determine the source of the increased plasma CGRP, the liver, spleen, small intestine, lungs, and heart were harvested, and tissue CGRP was assayed at 10 h after CLP in additional animals. Only the small intestine showed a significant increase in tissue levels of CGRP (by 129%, P < 0.05). Determination of portal vs. systemic levels of CGRP indicates that portal CGRP was 65.7 +/- 22.7% higher than the systemic level at 10 h after CLP, whereas portal CGRP in sham-operated rats was only 4.9 +/- 2.1% higher. Immunohistochemistry examination revealed that CGRP-positive stainings increased in the intestinal tissue but not in the liver at 10 h after the onset of sepsis. The distribution of CGRP stainings was associated with intestinal nerve fibers. These results, taken together, demonstrate that upregulation of CGRP occurs transiently during the progression of sepsis (at the late phase of the hyperdynamic sepsis), and the gut appears to be a major source of such an increase in circulating levels of this peptide.