Repair of orbital floor fractures using bioresorbable poly-L/DL-lactide plates

To assess the long-term clinical and radiologic findings after insertion of a bioresorbable polylactide plates P(L/DL)LA 70/30 implant (PolyMax) in the repair of orbital floor and wall defects, with special focus on stability and clinical signs of foreign-body reaction.

Development of novel melt-processable biopolymer nanocomposites based on poly(L-lactic acid) and WS2 inorganic nanotubes.

The use of tungsten disulphide inorganic nanotubes (INT-WS2) offers the opportunity to produce novel and advanced biopolymer-based nanocomposite materials with excellent nanoparticle dispersion without the need for modifiers or surfactants via conventional melt blending. The study of the non-isothermal melt-crystallization kinetics provides a clear picture of the transformation of poly(L-lactic acid) (PLLA) molecules from the non-ordered to the ordered state. The overall crystallization rate, final crystallinity and subsequent melting behaviour of PLLA were controlled by both the incorporation of INT-WS2 and the variation of the cooling rate. In particular, it was shown that INT-WS2 exhibits much more prominent nucleation activity on the crystallization of PLLA than other specific nucleating agents or nano-sized fillers. These features may be advantageous for the enhancement of mechanical properties and process-ability of PLLA-based materials. PLLA/INT-WS2 nanocomposites can be employed as low cost biodegradable materials for many eco-friendly and medical applications, and the exceptional crystallization behaviour observed opens new perspectives for scale-up and broader applications.

Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-l-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 μM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25–50% depending on whether one, two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2–4 nt. Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-l-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0.7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M. smegmatis, which does not export glutamine synthetase nor possess the poly-l-glutamate/glutamine (P-l-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.

Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2–5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.

l-Arginine-dependent suppression of apoptosis in Trypanosoma cruzi: Contribution of the nitric oxide and polyamine pathways

Until recently, a capacity for apoptosis and synthesis of nitric oxide (⋅NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated l-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with l-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite ⋅NO production. These effects were inhibited by the ⋅NO synthase inhibitor Nω-nitroarginine methyl ester (l-NAME). Exogenous ⋅NO limited DNA fragmentation but did not restore proliferation rates. Because l-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited l-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by l-arginine by means of (i) a ⋅NO synthase-dependent ⋅NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

L-arginine may mediate the therapeutic effects of low protein diets.

We have previously shown beneficial effects of dietary protein restriction on transforming growth factor beta (TGF-beta) expression and glomerular matrix accumulation in experimental glomerulonephritis. We hypothesized that these effects result from restriction of dietary L-arginine intake. Arginine is a precursor for three pathways, the products of which are involved in tissue injury and repair: nitric oxide, an effector molecule in inflammatory and immunological tissue injury; polyamines, which are required for DNA synthesis and cell growth; and proline, which is required for collagen production. Rats were fed six isocaloric diets differing in L-arginine and/or total protein content, starting immediately after induction of glomerulonephritis by injection of an antibody reactive to glomerular mesangial cells. Mesangial cell lysis and monocyte/macrophage infiltration did not differ with diet. However, restriction of dietary L-arginine intake, even when total protein intake was normal, resulted in decreased proteinuria, decreased expression of TGF-beta 1 mRNA and TGF-beta 1 protein, and decreased production and deposition of matrix components. L-Arginine, but not D-arginine, supplementation to low protein diets reversed these effects. These results implicate arginine as a key component in the beneficial effects of low protein diet.

Characterization of the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells as an NG-hydroxyl-L-arginine-nitric oxide adduct.

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

L-arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats

Nitric oxide (NO) is essential for normal function of the cardiovascular system. This study has determined whether chronic administration of L-arginine, the biological precursor of NO, attenuates the development of structural and functional changes in hearts and blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Uninephrectomized rats treated with DOCA (25 mg every 4th day sc) and 1% NaCl in the drinking water for 4 wk were treated with L-arginine (5% in food, 3.4 +/- 0.3 g.kg body wt(-1).day(-1)). Changes in cardiovascular structure and function were determined by echocardiography, microelectrode studies, histology, and studies in isolated hearts and thoracic aortic rings. DOCA-salt hypertensive rats developed hypertension, left ventricular hypertrophy with increased left ventricular wall thickness and decreased ventricular internal diameter, increased inflammatory cell infiltration, increased ventricular interstitial and perivascular collagen deposition, increased passive diastolic stiffness, prolonged action potential duration, increased oxidative stress, and inability to increase purine efflux in response to an increased workload. L-Arginine markedly attenuated or prevented these changes and also normalized the reduced efficacy of norepinephrine and acetylcholine in isolated thoracic aortic rings of DOCA-salt hypertensive rats. This study suggests that a functional NO deficit in blood vessels and heart due to decreased NO synthase activity or increased release of reactive oxygen species such as superoxide may be a key change initiating many aspects of the cardiovascular impairment observed in DOCA-salt hypertensive rats. These changes can be prevented or attenuated by administration of L-arginine.

L-arginine improves cardiovascular function in DOCA-salt hypertensive rats

No abstract

Novel three-component blends based on poly(L-lactide)

A significant number of poly a-ester homologues of poly(L-lactide) (PLLA) have been synthesized and used in miscibility studies together with conventional isomeric diacid-diol polyester variants, poly ß-esters (based on ß-hydroxybutyrate (HB) and ß-hydroxyvalerate (HV)), poly e-caprolactone (PCL), poly e-caprolactone copolymers (e.g. poly(L-lactide-co-caprolactone), and a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB), cellulose acetate propionate (CAP)). A combinatorial approach to rapid miscibility screening using 96-well plates and a uv-visible multi-wavelength plate reader has been developed enabling the clarity of PLLA-based multi-component blend films to be observed. Using these techniques and materials, the ternary phase compatibility diagrams of a range of three-component blend films was prepared, illustrating ranges of behavior varying from miscible blends giving rise to clear films to immiscible blends which are opaque. In this way, novel three-component blends of PLLA/CAB/PCL were developed which are miscible when the CAB content is more than 30%, PLLA less than 80% and PCL less than 60%.