Search

Cyclodextrins (CyDs) are water soluble, biocompatible cyclic oligosaccharides, made of ?-D-glucopyranose units joined by ?(1-4) linkages. They received considerable attention as carriers/solubilizers for drugs thanks to their ability to host lipophilic guests in their hydrophobic cavity. Various CyD-based nanoassemblies, loading drugs via non covalent interactions have been proposed as delivery platforms and some have been considered for preclinical studies. Water soluble CyD polymers can be prepared using crosslinking method based on polycondensation in the presence of a bifunctional agent, like citric acid, or epichlorohydrin. With epichlorohydrin a CyD content up to 70% w/w can be obtained for the water soluble polymers. Most interesting, CyD polymers are able to associate drugs much more efficiently than the monomeric CyD, to enhance the aqueous solubility of several guests and to incorporate two or more drugs or therapeutic agents contemporarily. They have been considered for the delivery of antibiotics and antitumorals and for the implementation of multimodal therapy. We will report on the successful loading in a polymeric ?CyD nanoparticle (NP) of the second-line tuberculosis drug Ethionamide together with a synthetic booster using a green approach and reaching high amount of dissolved drug. The NP loading both drug and booster revealed suitable for pulmonary spray application. Next, we will discuss encapsulation of Clofazimine, a riminophenazine derivative, known as antileprosy drug. It is known to be active against a wide spectrum of Gram-positive including Mycobacteria, Staphylococci, Streptococci, Enterococci and Listeria as well as MDR resistant strains. As Clofazimine is highly lipophilic, it tends to be deposited predominantly in fat tissues. During the last decade investigations, also in the field of nanotechnology, focused on alternative routes for improved CLZ delivery. We successfully loaded CLZ in sulfobutylether ?CyD polymers using a green method and obtained promising results against Staphylococcus Epidermidis, a microbe often found in hospital context.

Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functionsviacovalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis.The isolation and analysis of endogenous poly (ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteinsin vitroare also present under physiologic conditionsin vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.

Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2-/- mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.

The ability of an engineering plastic material to maintain properties at increased temperature is critical in many applications. Here is discussed how recent developments in catalyst technology have led to the ability to produce syndiotactic polystyrene (See Figure), the regularly alternating arrangement of pendant phenyl groups giving rise to a crystalline material with well-structured spherulitic morphology and improved heat performance.

The creep performance of a material is one of the main criteria currently used to assess the long-term performance of thermoplastic composites. In a wide number of applications, including electrical connectors and various automotive applications, dimensional stability at elevated temperatures (i.e., short-term creep performance) is an essential design requirement. In many of these applications the material can be exposed to elevated temperatures and high applied stresses as part of the fabrication process or in the end-use application. The elevated temperature creep behavior of 30%, and 40% glass-filled syndiotactic polystyrene, 30% glass-filled poly(butylene terephthalate), 30% glass-filled poly(phenylene sulfide), and a 30% glass-filled liquid crystalline polymer has been evaluated. The creep behavior for each of the materials has been modeled using the formulation proposed by Findley, Kholsa, and Petersen (FKP). The FKP model was found to provide a good description of the creep performance of these glass-filled materials. The parameters in the FKP model were evaluated for each of the materials as a function of temperature and applied stress. Comparison of the predicted short-term creep behavior using the FKP model with experimental data has shown that the predicted creep behavior is within ±10% of the measured value over the temperature and applied stress ranges examined. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67:1177–1183, 1998

ABSTR.15

ABSTR. P.397

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

In the present paper, we investigated the relationship between the growth inhibitory effects of recombinant interferon-alpha2b (rIFN-alpha2b) and poly (ADPR) polymerase-1 (PARP-1) activity in the human squamous KB cancer cell line. Growth inhibition of the KB cells mediated by 1000 IU/ml of rIFN-alpha2b was accompanied by a transient rise in PARP-1 specific activity 24 h after rIFN-alpha2b treatment, confirmed by both the increase of intracellular poly (ADP-ribose) content and the PARP-1 auto-modification level. At longer times of incubation, the onset of apoptosis accompanied KB cell growth inhibition, as demonstrated by both flow cytometry and western-blotting analysis showing an 89 kDa apoptotic fragment of PARP-1. Moreover, pretreatment of the cells with the PARP-1 inhibitor, 3-aminobenzamide (3-ABA), at non-cytotoxic concentrations (1 mM), reduced the cell-growth inhibition, cell-cycle perturbation and apoptosis caused by rIFN-alpha2b. Taken together, these results strongly suggest that PARP-1 may be directly involved in the effects of rIFN-alpha2b in the KB cancer cell line.

ABSTR. IX

ABSTR.39A

Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.

Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing. Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis. In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism. In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53- and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions.

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.

We have found that human cells respond to treatment with an alkylating carcinogen by producing a specific, highly conserved array of poly(ADP-ribose) molecules. Using in situ radiolabeling and boronate affinity chromatography, we have been able to isolate ADP-ribose polymers from living cells and to quantify individual molecular size classes on high resolution polyacrylamide gels. Despite carcinogen-induced changes in poly(ADP-ribose) turnover, the relative frequency of linear polymers was strictly conserved. However, the abundance of branched polymers with high affinity for histones increased 2.6-fold. These polymers were degraded more slowly than linear polymers. Our results indicate that the poly(ADP-ribose) molecules involved in the shuttling of histones on DNA in vitro also play a role in the repair of DNA damage in vivo.

After Zn2+ finger-mediated binding to a DNA break, poly(ADP-ribose) polymerase becomes automodified with long polymers of ADP-ribose. These nucleic acid-like polymers may facilitate DNA repair by noncovalently interacting with neighboring proteins. Using a novel screening technique, we have identified histones as the predominant poly(ADP-ribose)-binding species in human keratinocytes, rat hepatocytes, frog eggs, and yeast. Polymer binding is confined specifically to the histone domains responsible for DNA condensation, i.e. histone tails. Our results indicate that polymers of ADP-ribose are targeted to sites of DNA strand breaks by poly(ADP-ribose) polymerase and subsequently function to alter chromatin conformation through noncovalent interactions with histone tails.

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.

ADP-ribosylation of a specific basic protein has been investigated in isolated intact bull testis nuclei incubated with NAD + . The electrophoretic mobility, molecular weight and amino-acid composition of the purified bull testis specific protein are similar to those of rat testis protein. About 1–5% of the total radioactivity incorporated in the 20% acid-insoluble fraction was associated with testis protein and was identified as ADP-ribose.

In "Proceedings of the 7th Intern. Symp. on ADP-ribosylation Reactions"

A number of new polymers have been studied. They include polymers obtained by chain reaction polymerization of olefins and epoxides with functional groups where the polymerizable group was separated from the functional group by an inert spacer group, the preparation of head-to-head polymers, and the preparation of optically active polymers based on the macromolecular asymmetry.

Poly(1,1-dimethylpropane) was synthesized by Wurtz coupling polymerization. The polymer was of moderate molecular weight, about 50% crystalline, had a melting temperature of 87°C and a glass transition temperature of −17°C. The polymer was essentially the same material as that obtained by cationic hydride transfer polymerization of 3-methylbutene-1 at very low temperatures, or by cationic ring opening polymerization of 1,1-dimethylcyclopropane. Poly(1,1-dimethylpropane) obtained by the Wurtz reaction was obtained in only low yield; a substantial part of the total reaction product was 1,1-dimethylcyclopropane. Poly(1,1-dimethylpropan) wurde mittels der Wurtzschen Reaktion synthetisiert. Das erhaltene Polymer hat nur masige relative Molekulmasse, ist zu etwa 50% kristallin und hat eine Schmelztemperatur von 87°C sowie eine Glasubergangstemperatur von −17°C. Das Polymer ist im wesentlichen das gleiche Material, das durch kationische Hydrid-transfer-Polymerisation von 3-Methyl-buten-1 bei niedrigen Temperaturen oder durch kationische Ringoffnungs-polymerisation von 1,1-Dimethylcyclopropan erhalten wird. Poly(1,1-dimethylpropan) wurde nach der Wurtzschen Synthese nur in niedriger Ausbeute erhalten; ein wesentlicher Teil des Reaktionsproduktes war 1,1-Dimethylcyclopropan.

Summary1. Preparation of 7 substituted imidazoles has been described. 2. Anticoccidial activity was demonstrated for imidazole-4-carboxamide and certain related compounds. 3. The most potent compound was 4,5-imidazoledicarboxamide (glycarbylamide). 4. Replacement of one or both carboxamides in the 4.5 positions on the imidazole ring reduced or completely eliminated the anticoccidial activity of glycarbylamide. Substitutions at the 1 or 2 positions on the imidazole ring produced compounds of less activity than glycarbylamide.

Summary1. The data presented show that nithiazide [1-ethyl-3-(5-nitro-2-thi-azolyl) urea | was more potent and less toxic than 2-amino-5-nitrothiazole when fed to turkeys with enterohepatitis. 2. Nithiazide was most effective when administered prior to infection or beginning 3 to 7 days after infection. It prevented mortality from enterohepatitis produced either by oral exposure to cecal worm eggs or by rectal inoculation of Histomonas meleagridis cultures. Nithiazide may be administered in the feed or water and is equally effective in either form. 3. The therapeutic feeding of rations containing 0.05% nithiazide, starting 3 days after infection, prevented mortality from infectious enterohepatitis which was lethal for 73% to 83% of the untreated control turkeys.