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IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Background. While anabolic hormone deficit is a common finding in heart failure with reduced ejection fraction (HFrEF), few data are available in heart failure with preserved ejection fraction (HFpEF). Methods. Blood samples were collected for metabolic (total cholesterol, HDL cholesterol, LDL cholesterol, creatinine, and glucose) and hormonal (IGF-1, DHEA-S, TSH, fT3, fT4, and T) determination, comparing 30 patients with HFpEF and 20 patients with HFrEF. Total antioxidant capacity was evaluated by using the spectrophotometric method using the latency time in the appearance of the radical species of a chromogen (LAG, sec) as a parameter proportional to antioxidant content of the sample. Echocardiographic parameters were also assessed in the two groups. Results. A high prevalence of testosterone (32% in HFrEF and 72% in HFpEF, p<0.05) and DHEA-S deficiencies was observed in HFpEF patients. Echocardiographic parameters did not correlate with hormone values. A significant direct correlation between T (r2 = 0.25, p<0.05) and DHEA-S (r2 = 0.19, p<0.05) with LAG was observed only in HFpEF. Conclusion. Anabolic hormone deficiency is clearly shown in HFpEF, as already known in HFrEF. Although longitudinal studies are required to confirm the prognostic value of this observation, our data suggest different mechanisms in modulating antioxidants in the two conditions, with possible therapeutic implications.

Industrial hemp (Cannabis sativa) is traditionally cultivated as a valuable source of fibers and nutrients. Multiple studies also demonstrated antimicrobial, anti-proliferative, phytotoxic and insecticide effects of the essential oil from hemp female inflorescences. On the other side, only a few studies explored the potential pharmacological application of polar extracts from inflorescences. In the present study, we investigated the water extract from inflorescences of industrial hemp Futura 75 variety, from phytochemical and pharmacological point of view. The water extract was assayed for phenolic compound content, radical scavenger/reducing, chelating and anti-tyrosinase effects. Through an ex vivo model of toxicity induced by lipopolysaccharide (LPS) on isolated rat colon and liver, we explored the extract effects on serotonin, dopamine and kynurenine pathways and the production of prostaglandin (PG)E2. Anti-proliferative effects were also evaluated against human colon cancer HCT116 cell line. Additionally, antimycotic effects were investigated against Trichophyton rubrum, Trichophyton interdigitale, Microsporum gypseum. Finally, in silico studies, including bioinformatics, network pharmacology and docking approaches were conducted in order to predict the putative targets underlying the observed pharmacological and microbiological effects. Futura 75 water extract was able to blunt LPS-induced reduction of serotonin and increase of dopamine and kynurenine turnover, in rat colon. Additionally, the reduction of PGE2 levels was observed in both colon and liver specimens, as well. The extract inhibited the HCT116 cell viability, the growth of T. rubrum and T. interdigitale and the activity of tyrosinase, in vitro, whereas in silico studies highlighting the inhibitions of cyclooxygenase-1 (induced by carvacrol), carbonic anhydrase IX (induced by chlorogenic acid and gallic acid) and lanosterol 14-&alpha, demethylase (induced by rutin) further support the observed pharmacological and antimycotic effects. The present findings suggest female inflorescences from industrial hemp as high quality by-products, thus representing promising sources of nutraceuticals and cosmeceuticals against inflammatory and infectious diseases.

ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co-purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.

Tricholosporum goniospermum (Bres.) Guzm&aacute, n ex T.J. Baroni is an excellent edible mushroom whose compounds and biological properties are still unknown. In this study, n-hexane, ethyl acetate and methanol extracts from fruiting bodies and liquid-cultured mycelia were compared for the analysis of phenolic compounds, the evaluation of scavenger (DPPH, ABTS) and reducing (CUPRAC, FRAP) activities, and the enzyme inhibition of &alpha, amylase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase. Additionally, T. goniospermum extracts were evaluated for antibacterial and antimycotic activities against Gram+ and Gram&minus, bacteria, and clinical yeast and fungal dermatophytes. Finally, based on the extract content in phenolic compounds, in silico studies, including the docking approach, were conducted to predict the putative targets (namely tyrosinase, lanosterol-14-&alpha, demethylase, the multidrug efflux system transporters of E. coli (mdtK) and P. aeruginosa (pmpM), and S. aureus &beta, lactamase (ORF259)) underlying the observed bio-pharmacological and microbiological effects. The methanolic extract from mycelia was the richest in gallic acid, whereas the ethyl acetate extract from fruiting bodies was the sole extract to show levels of catechin. Specifically, docking runs demonstrated an affinity of catechin towards all docked proteins, in the micromolar range. These in silico data are consistent, at least in part, with the highest activity of ethyl acetate extract as an antimicrobial and anti-tyrosinase (554.30 mg KAE/g for fruiting bodies and 412.81 mg KAE/g for mycelia) agent. The ethyl acetate extracts were also noted as being the most active (2.97 mmol ACAE/g for fruiting bodies and 2.25 mmol ACAE/g for mycelia) on &alpha, amylase. BChE inhibitory activities varied from 2.61 to 26.78 mg GALAE/g, while the tested extracts were not active on AChE. In conclusion, all mushroom extracts tested in this study had potent antimicrobial activities. Particularly, among the tested extracts, the ethyl acetate extract showed the highest efficacy as both an antimicrobial and anti-tyrosinase agent. This could be related, albeit partially, to its content of catechin. In this regard, the bioinformatics analyses showed interactions of catechin with tyrosinase and specific microbial proteins involved in the resistance to chemotherapeutic drugs, thus suggesting innovative pharmacological applications of T. goniospermum extracts.

In the present study, Passiflora foetida extracts characterized by different polarities were studied for their phytochemical profile, enzyme inhibitory, and antioxidant potentials. In silico, in vitro and ex vivo studies were also carried out on methanol and water extracts for predicting pharmacokinetics and pharmacodynamics. In this regard, neuronal HypoE22 cells, isolated mouse skin tissues, and pathogen dermatophytes strains were exposed to extracts. Emphasis was given to the preventing effects induced by the extracts on hydrogen peroxide-induced alterations of prostaglandin E2 (PGE2), l-dopa, and serotonin. Chemical analysis revealed the presence of similar compounds in infusion and methanolic extracts. The ex vivo studies also showed protective skin properties by P. foetida water and methanol extracts, as evidenced by the decrease of hydrogen peroxide-induced PGE2 level. Additionally, the blunting effects on hydrogen peroxide-induced l-dopa levels are consistent with the anti-tyrosinase effect exerted by both extracts. In silico studies demonstrated the affinity of extracts&rsquo, phytochemicals, namely apigenin, chrysoeriol, loliolide, luteolin, quercetin, and vitexin, towards cyclo-oxygenase-2 and tyrosinase. Finally, microbiological tests demonstrated the efficacy of P. foetida methanol and water extracts as anti-mycotic agents against Trichophyton and Arthroderma species, involved in skin inflammation. Hence, P. foetida L. extracts could represent potential sources of pharmaceuticals and nutraceuticals.

In this study, ethyl acetate, methanol, and water extracts of Bersama abyssinica (Melianthaceae) stem bark were screened for enzyme inhibitory and antioxidant properties. The water extract possessed the highest concentration of phenols (230.83 mg gallic acid equivalent/g extract), while the methanol extract was rich in flavonoids (75.82 mg rutin equivalent/g extract), and the ethyl acetate extract possessed the highest amount of saponins (97.37 mg quillaja equivalent/g). The aim of this study was to investigate the antiproliferative effects against the human colon cancer HCT116 cell line challenged with serotonin (5-HT) as a stimulating-proliferation factor. The level of HCT116 cell-deriving pool of kynurenic acid (KA) was also assessed. The UHPLC results confirmed the presence of 58, 68, and 63 compounds in the ethyl acetate, methanol, and water extracts, respectively. Mangiferin, vitexin and its isomer isovitexin were tentatively identified in all extracts and KA (m/z 190.05042 [M?H]+) was also tentatively identified in the methanol and water extracts. The methanol extract (1464.08 mg Trolox equivalent [TE]/g extract) showed the highest activity in the CUPRAC assay, whereas the water extract (1063.70 mg TE/g extract) showed the highest activity with the FRAP technique. The ethyl acetate extract was the most active acetylcholinesterase (4.43 mg galantamine equivalent/g extract) and ?-glucosidase (mmol acarbose equivalent /g extract) inhibitor. The water extract was able to inhibit 5-HT-stimulated viability of HCT116 cells, and blunt 5-HT-induced reduction of cell-deriving KA. The scientific data generated in this study provide baseline data regarding the biological properties of B. abyssinica stem bark, highlighting its potential use for the development of new pharmaceutic and cosmetic agents. © 2020 by the authors. Licensee MDPI, Basel, Switzerland., FAR 2019, Funding: The study was supported by the Italian Ministry of University funds (FAR 2019) granted to Proff. Giustino Orlando, Annalisa Chiavaroli, and Claudio Ferrante.

Coronilla species, belonging to the Coronilla genus (Fabaceae), have long been used in traditional medicine for treating cold, diabetes, pain, and as cardiotonics. The goal of the present study was to explore the phytochemical composition and pharmaco-toxicological properties of C. minima. In this regard, phenolic content, scavenging/reducing properties and antimicrobial activity toward pathogen bacterial (Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus) and fungal strains (Candida albicans, C. tropicalis, Aspergillus tubigensis and A. minutus) were investigated. Extract effects on human colon cancer HCT116 cell viability were also assayed. Finally, a bioinformatics approach was conducted with the aim to identify putative microbial and human protein targets underlying antibacterial, antimycotic, and antiproliferative effects. Phytochemical investigation suggested that water extract is richer in terms of total flavonoid and phenol content, whereas the hydroalcoholic extract was revealed to be more potent as antioxidant agent. According to bioinformatics analysis, the antibacterial activity of the hydroalcoholic extract could be related to its content in resveratrol. The presence of resveratrol could also explain the hydroalcoholic extract efficacy in reducing HCT116 cell viability. In conclusion, the present study represents the first phytochemical and bio-pharmacological investigation about C. minima. Like other plants belonging to the Fabaceae family, C. minima revealed a good source of resveratrol, which could explain, albeit partially, the efficacy of the hydroalcoholic extract as antimicrobial, antioxidant, and antiproliferative agent.

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.

The stem bark (SB) of Piptadeniastrum africanum (PA) has been extensively used in African traditional medicinal systems. However, there is a dearth of scientific information regarding its possible activity in the management of type II diabetes, Alzheimer&rsquo, s disease, and skin hyperpigmentation disorders. This study therefore attempted to elucidate the in vitro inhibitory action of ethyl acetate, methanol, and water extracts of P. africanum stem bark (PA-SB) on &alpha, amylase, &alpha, glucosidase, acetylcholinesterase, butyrylcholinesterase, and tyrosinase. Cell viability, catecholamine, and 3-hydroxykynurenine levels of hypothalamic HypoE22 cells exposed to PA-SB extracts were also investigated. The phytochemical profiles of the extracts were determined by high performance liquid chromatography (HPLC) and antioxidant properties were investigated. Saponin (867.42 mg quillaja equivalent/g) and tannin (33.81 mg catechin equivalent/g) contents were higher in the methanol extract. Multiple dihydroxy-trimethoxy(iso)flavone isomers, loliolide, eriodictyol, naringenin, luteolin, chrysoeriol, apigenin, and liquiritigenin, were characterized from PA-SB extracts using HPLC. The methanol extract of PA-SB showed highest inhibitory activity against acetylcholinesterase (4.88 mg galantamine equivalent (GALAE)/g extract), butyrylcholinesterase (5.37 mg GALAE/g extract), and tyrosinase (154.86 mg kojic acid equivalent/g extract) while &alpha, glucosidase was effectively inhibited by the ethyl acetate extract (15.22 mmol acarbose equivalent/g extract). The methanol extract of PA-SB also showed potent antioxidant properties (493.87, 818.12, 953.07, and 732.19 mg Trolox equivalent/g extract, for 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2&prime, azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays, respectively). PA-SB extracts exhibited antioxidant activity and promising inhibition against key enzymes related to type II diabetes, Alzheimer&rsquo, s disease, and skin hyperpigmentation disorders. Additionally, all extracts were able to contrast hydrogen peroxide-induced oxidative stress, in HypoE22 cells, thus restoring basal catecholamine and 3-hydroxykinurenine levels, whereas only methanol and water extracts stimulated basal dopamine release. Overall, data from the present study contribute to the biological assessment of P. africanum that appears to be a promising source of natural compounds with protective and neuromodulatory effects.

Background The altered composition of the gastrointestinal (GI) microbiota, known as dysbiosis, can induce and modulate the systemic inflammation, through microbial translocation and T-cell activation, in several immuno-mediated diseases, such as inflammatory bowel disease (IBD), HIV infection, and ankylosing spondylitis. Objectives In a cohort of 85 patients with inflammatory bowel disease-associated spondyloarthritis (SpA/IBD), we assessed gut bacterial infiltration and intestinal damage. In systemic circulation, GI epithelial damage, microbial translocation and immune system activation were assessed with intestinal-fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), soluble CD14 (sCD14), respectively. Moreover, the in vitro activity of the latter two was evaluated on osteoblast cells. Methods I-FABP, LPS, sCD14, sclerostin (SOST) and anti-SOST antibodies (anti-SOST-IgG) were assayed with ELISAs. LPS and sCD14 were used in vitro to stimulate the MG63 human osteoblast-like cell line. Occludin, claudin-1, claudin-4, and the presence of bacteria were assessed, respectively, by real-time-PCR analysis and immunohystochemical staining of the ileum. Results Bacteria were detectable in the ileal epithelium of IBD patients, but only in SpA/IBD they were associated with epithelial damage and downregulation of occludin, claudin-1 and claudin-4 (figure 1A-B). The serum levels of I-FABP, LPS and sCD14 resulted significantly higher in axial (187.9, 14.03, and 26.97, respectively) and peripheral SpA/IBD (130.3, 11.55, and 18, respectively) than in IBD patients (IFABP 43.65, p In the SpA/IBD cohort, SOST was weakly correlated with I-FABP (r=-0.2683), LPS (r=-0.3063) and sCD14 (r=-0.3075), and anti-SOST-IgG with LPS (r=- 0.3959) and sCD14 (r=-0.3414). Moreover, sCD14 showed significant correlation with I-FABP (r=0.3316) and LPS (r=0.5649). In vitro , LPS, but not sCD14, significantly induced SOST expression through the upregulation of both Wnt3a and Wnt5a and the downregulation of the b-catenin proteins (figure 1D). On the opposite, the combination of LPS and sCD14 downregulated SOST expression through the upregulation of ERK1/2 and b-catenin protein (figure 1D). Conclusions The role of gut inflammation and microbial translocation in the onset of arthritis in IBD patients are still under investigation. We have demonstrated that in SpA/IBD there is a significant bacterial infiltration of the ileal tract, associated with the downregulation of tight-junctions’ proteins (occludin, claudin-1 and claudin-4) and epithelial damage, that cause microbial translocation and higher plasma levels of I-FABP, LPS, and sCD14. LPS and sCD14, thus, could trigger a complex systemic inflammatory response acting on several biochemical pathways, linking the immune system (anti-SOST-IgG) and the bone (SOST). Disclosure of Interest None declared

Whole-genome sequencing was used to examine a persistent Enterococcus faecium bacteremia that acquired heteroresistance to three antibiotics in response to prolonged multidrug therapy. A comparison of the complete genomes before and after each change revealed the emergence of known resistance determinants for vancomycin and linezolid and suggested that a novel mutation in fabF , encoding a fatty acid synthase, was responsible for daptomycin nonsusceptibility. Plasmid recombination contributed to the progressive loss of vancomycin resistance after withdrawal of the drug.

Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival., ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.

Influenza B virus (IBV) causes annual influenza epidemics around the world. Here we use an in vivo plasmablast enrichment technique to isolate a human monoclonal antibody, 46B8 that neutralizes all IBVs tested in vitro and protects mice against lethal challenge of all IBVs tested when administered 72 h post infection. 46B8 demonstrates a superior therapeutic benefit over Tamiflu and has an additive antiviral effect in combination with Tamiflu. 46B8 binds to a conserved epitope in the vestigial esterase domain of hemagglutinin (HA) and blocks HA-mediated membrane fusion. After passage of the B/Brisbane/60/2008 virus in the presence of 46B8, we isolated three resistant clones, all harbouring the same mutation (Ser301Phe) in HA that abolishes 46B8 binding to HA at low pH. Interestingly, 46B8 is still able to protect mice against lethal challenge of the mutant viruses, possibly owing to its ability to mediate antibody-dependent cellular cytotoxicity (ADCC)., Influenza B virus (IBV) co-circulates with influenza A virus to cause annual epidemics. Here, Chai et al. isolate a human monoclonal antibody that binds to a conserved epitope in the viral HA protein, neutralizes IBV strains in vitro, and protects mice against IBV infection.

Human cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients.

Different syntheses of ordered condensation polymers from nonsymmetric monomers are reviewed. For several classes of polymers, the approach exploiting reactivity differences between the functional groups of the nonsymmetric monomers (ordered polymers from in situ generated intermediates) is compared with that where the relevant intermediates are used as symmetrical preformed and/or isolated precursors for the ordered polymers.

Tailored diblock copolymers of rigid poly(p-benzamide) (PBA) and flexible poly(ethylene glycol) (PEG) were prepared using two different polymerization schemes: (i) the one-pot two-step scheme of the Yamazaki reaction including benzoyl-terminated PBA and a PEG methyl ether modified by p-aminobenzoylation; (ii) the polymerization of p-aminobenzoic acid in the presence of the above NH 2 -terminated PEG. The former method yields a copolymer coexisting with a large amount of PBA homopolymer. The latter method yields a copolymer with a reduced and controlled amount of unblocked PBA. The fraction of flexible segments in the copolymers varied between ca. 0.60 and ca. 0.40. In solutions of N,N-dimethylacetamide/LiCl, the solubility of the flexible homopolymer is larger at low LiCl content when the solubility of rigid PBA is, instead, negligeably small. However, large quantities of the latter are solubilized at low LiCI content, provided the copolymer is also present. The order of increasing solubility of the two components is reversed at high LiCl content. Micellar-type organizations of the amphiphilic copolymer with the rigid homopolymer are postulated. A lyotropic mesophase appears at a copolymer volume fraction v p 1 ca. 0.06 and it is fully formed at v p a ca. 0.08. The evolution of the structurization from diluted solutions to the mesophase and to the solid state is briefly considered.

The mmr gene, cloned from Mycobacterium tuberculosis , was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [ 3 H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP.

Copolymers were prepared by the condensation of rigid benzoyl-terminated poly(p-benzamide) and flexible anilino-terminated poly(m-benzamide) prepolymers. The use of two monomers of the AB type and of end-capped prepolymers resulted in the formation of diblock molecules uncontaminated by triblock or multiblock sequences. Moreover, selective extraction techniques in N,N-dimethylacetamide (DMAc) with varying amounts of LiCI resulted in the complete elimination of unreacted prepolymers, as evidenced from material balance and 'H NMR data. The above extraction technique also allowed a fractionation of the copolymers in terms of their rigid/flexible compositional distribution ratio. The molecular weight-intrinsic viscosity dependence of poly(m-benzamide) determined in a nonaggregating solvent using light scattering yielded a persistence length of 8 A, suggesting a relatively large chain flexibility. A tailored copolymer with a fraction β of flexible residues = 0.70 was fractionated, determining the solubility, the viscosity, and the critical composition for mesophase formation in DMAc/LiCl and in 96% H 2 SO 4 solutions. The results, in line with those previously reported for a related system, reveal that the addition of the flexible segment has a little effect on the critical concentration of poly(p-benzamide), whereas the biphasic gap is strongly reduced. Moreover, the solubility of the copolymer is substantially increased over that of the rigid homopolymer. The net result is an unexpected widening of the range of stability of the pure mesophase, with significant implications from both a fundamental and an applied standpoint.

We have recently reported high clinical activity against advanced colorectal cancer of a regimen-alternating bolus FUra, modulated by methotrexate (MTX), and continuous infusion FUra, modulated by 6-s-leucovorin (6-s-LV). Considering the low toxicity of the bolus part of this regimen and our recent in vitro finding of a strong synergism between bolus FUra and natural-beta-IFN (n-beta-IFN), this cytokine was incorporated in the bolus part of our treatment programme. Fifty-six patients with untreated, advanced, measurable colorectal cancer were treated with two biweekly cycles of FUra bolus (600 mg m(-2)), modulated by MTX (24 h earlier, 200 mg m(-2)), and n-beta-IFN (3 x 10(6) IU i.m. every 12 h, starting at the time of FUra administration for four doses), alternating with a 3-week continuous infusion of FUra (200 mg m(-2) daily), modulated by 6-s-LV (20 mg m(-2) weekly bolus). After a 1-week rest, the whole cycle (8 weeks) was repeated if indicated. A total of 5 complete and 17 partial responses were obtained (response rate, 41%; 95% confidence limits, 28-55%) in 54 assessable patients. After a median follow-up time of 36 months, five patients are still alive. Overall, the median time to treatment failure was 6.4 months. The median duration of survival was 15.0 months. There was one treatment-related death after a course of MTX --> bolus FUra/n-beta-IFN and grade III-IV toxicity occurred in 18% of the patients. As the addition of n-beta-IFN results in high toxicity, whereas the efficacy seems to be similar to that of the same regimen without the cytokine, our groups are currently randomizing the original regimen, without IFN, against standard modulated bolus FUra.

The determination of single-chain properties for heterogeneous molecules such as a rigid-flexible block copolymer requires the use of solvents capable of reducing selective interactions involving the blocks and the diluent. Molecular dispersion is displayed by a two-block copolymer of benzoyl-terminated poly(p-benzamide) (poly(imino-1,4-phenyl-enecarbonyl) and anilino-terminated poly(m-phenyleneisophthalamide) in 96 wt.-% sulfuric acid. For a series of such copolymers, intrinsic viscosities were measured avoiding degradation effects due to H 2 SO 4 . The intrinsic viscosity ([η]) of the copolymers was found to decrease with increasing length of the flexible block, but [η] remained larger than the value corresponding to an equimolar blend of rigid and flexible homopolymers. These results are explained by theoretical considerations within the framework of the hydrodynamic behavior of single rodlike and single coiled macromolecules.

Therapeutic response to angiotensin-converting enzyme (ACE) inhibitors was reported to be better related to tissular than to circulating levels of ACE inhibition, especially during chronic therapy. We studied the relations between plasma concentrations of angiotensin I (AI), plasma renin activity (PRA), angiotensin II (AII), and aldosterone (by radioimmunoassay, RIA) and levels of serum and tissue ACE activities during acute and chronic quinapril administration in rats. Forty-eight male Wistar rats received quinapril by gavage for either I day (n = 24) or 15 days (n = 24) at different doses (control, 0.1, 1, and 10 mg/kg/day ; 6 rats at each dose). Plasma hormonal parameters, serum, and tissue (lung, heart, and aorta) ACE activities were measured 3 h after the last gavage. Significant dose-dependent inhibitions of serum and lung ACE during acute and chronic treatments were observed (p < 0.05). Degrees of serum and heart ACE inhibition (at 0.1 mg/kg/day) were significantly lower with chronic than with acute treatment (p < 0.05). Degree of inhibition in lung, which represents the main source of total ACE, was similar during acute and chronic treatments. Among plasma hormonal parameters, plasma AI was correlated to PRA and showed the best correlation with ACE inhibition. After logarithmic transformation, log AI was significantly correlated to ACE activity in lung during chronic treatment (r = -0.85, p < 0.05). This parameter may provide a useful index for ACE inhibitor dosage adjustment during chronic therapy.

Only a few bacterial and fungal species present in a soil extract grew and survived on painted canvases prepared utilizing the materials and following the recipes traditionally used for paintings. When pure cultures of these microorganisms were used to contaminate such canvases, microbial growth and survival were even more reduced. On the other hand, on this substrate, the bacteria isolated from a heavily contaminated XVIth century fresco grew to a limited extent, but remained viable for a much longer period of time. Unlike the bacteria isolated from soil, those isolated from the fresco were able to hydrolyse cellulose and proteins. It is thus possible that the capacity to utilize these macromolecules may favour microbial survival on paintings. Furthermore, the addition of phosphate strongly stimulates growth of all microbial species indicating that this anion is the limiting nutrient factor.

New types of polysaccharidic assemblies based on chitosan and uncharged or charged derivatives of scleroglucan are presented. Macroscopic assemblies having a spherical skin-like shape were prepared by controlled mixing of two polyelectrolyte solutions. Hydrogels based exclusively on the above natural derivatized polysaccharides crosslinked via simple chemical processes, avoiding the use of extraneous reticulating agents and organic solvents, are also described. These biocompatible materials may, eventually, be used in drug release formulations or for the incapsulation of bioactive macromolecules. The interesting possibility exists to study the micelle formation of amphiphiles within the spherical skins.

Aqueous solutions of (hydroxypropyl) cellulose, which can be either isotropic (dilute regime) or cholesteric (concentrated regime) at room temperature, exhibit reversible phase separation upon heating above ∼37 °C. The biphasic system does not gel and includes a concentrated phase having the appearance of an emulsion of poorly organized micro aggregates, and a dilute solution from which the concentrated phase cannot be completely separated. Ellipticity data suggest the onset of cholesteric order at temperatures slightly below those at which isotropic solutions undergo phase separation. This hint of the formation of the mesophase is detected at concentrations unusually low (∼0.4% v/v). The conjugation of the two phases was evidenced by determination of the individual volumes and concentrations. The pseudo phase diagram includes boundaries for the high-temperature biphasic region which are not readily interpreted in terms of a heat-induced crystallization process. It is suggested that the primary event controlling phase separation is the driving force for the formation of a mesophase in a solvent which becomes poorer upon increasing temperature (inverted wide region of the theoretical diagram). The development of well-organized liquid crystalline domains, and their possible evolution toward a crystalline state, may be greatly slowed down by kinetic effects.

Complexes formed by DNA and lipid mixtures have great interest for the assessment of self-assembling mechanisms in open biological systems. X-ray diffraction has revealed that minor alterations of the cationic/neutral lipid composition produced major alterations to the liquid crystalline structure and the hydration of the complexes. We have extended to these systems by an approach based on the identification of a fundamental repeating unit that grows according to the general principles of supramolecular polymerisation and liquid crystallinity. Structural reorganisations that optimise the electrostatic and hydrophobic compensation are enhanced by competition between the rigidity of the polyelectrolyte and the cohesion of the lipid assembly. The non-hydrated hexagonal structure revealed by X-ray examination is represented by a dendritic-type supramolecular polymerisation of DNA units decorated by the aliphatic tails of dissociated liposomes. An increase in the cationic/neutral lipid component ratio enhances the stability of planar bilayers, favouring the formation of the partly hydrated lamellar structure revealed by X-ray diffraction

To elucidate the role of fixed charge and chain rigidity on the assembly of amphiphilic compounds over a polymer substrate, we studied the interaction between sodium dodecyl sulfate (SDS) and crystalline or soluble collagen. Shrinkage temperatures and the equilibrium degree of swelling were measured for crosslinked tendons under isoelectric (pH=6.0) conditions or in acid (pH=2.5) solutions. Viscosities and solubilities were measured for soluble collagen to construct pseudo phase diagrams delimiting the field of stability of the helical, random coiled, and crystalline forms. Under isoelectric conditions, increasing concentrations of SDS (up to ≃0.1 M) cause a depression of transformation temperatures which is much larger than that observed with salts, with aliphatic alcohols, or with sodium methyl sulfate

Dilute solutions of fractions of two polyamides (PA-2 and PA-4) having a phenyl substituent on every fourth ring are studied. With respect to their unsubstituted analogues, poly(p-benzamide) (PBA) and poly(p-phenylenediamine-terephthalic acid) (PPTA) respectively, PA-2 and PA-4 show increased solubility in organic solvents and smaller persistence length, q. It is concluded that even one phenyl substituent on every four rings has a significant effect in making the chain more flexible. The chains still retain the ability to form a mesophase in concentrated solutions

The thermodynamic framework and recent molecular descriptions of protein-salt interactions are critically reviewed. A reanalysis of earlier and recent data describing the role of salts on the thermal stability of collagen I over a wide range of concentration evidences the complex encroachment of charge-dependent and charge-independent interactions. Charge-independent interactions, operationally quantified by dilution parameters and association constants, are found to be the overriding factor for the large stabilization observed with nonbinding salts and for the large destabilization observed when strong salt binding occurs. Charge-dependent interactions, namely screening and selective ion adsorption, are instead the prevailing components at low ionic strength and nonisoelectric pH.

Intrinsic bent DNA sites were identified in the 4289 bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides -6753, -5433, -5133 and -4757. Sequence analysis showed that these bends are composed of 2-6 bp dA.dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289 bp sequence revealed low helical stability (DeltaG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.

Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids.

The therapeutic activity and tolerability of sobrerol, a muco-active agent, were evaluated in 30 children (aged 5–10 years) with secretory otitis media. The drug was administered once daily by inhalation for 10 days consecutively at a dose of 40 mg/3 ml. Clinical assessments showed a significant improvement in the objective measures together with good tolerability. Moreover, sobrerol was shown to improve impedance values; this is an important aspect of the modifications induced by this drug in diseases involving the ear.

A novel extension of the Yamazaki reaction is used to prepare block copolymers having rigid blocks of poly-(p-benzamide) (PBA) and semiflexible blocks of polyamide-hydrazide. A PBA prepolymer having M ≅ 10,000 was synthesized by the usual Yamazaki reaction using triphenylphosphite. As previously reported, higher-molecular-weight PBA could be obtained using 4-N-(4′-aminobenzamido)benzoic acid containing a preformed amide linkage. Addition of p-aminobenzhydrazide and terephthalic acid then led to formation of the polyamide-hydrazide blocks using as the active reactant the diphenylphosphite formed as a by-product in the first polymerization. Evidence that a block copolymer is produced includes an increase in inherent viscosity during the second step, differences in the solubility of the copolymer compared to the homopolymers, and comparison of the phase diagram of the block copolymer in N-methylpyrrolidone having 4% added LiCl with those of a random copolymer, and of mixtures of the two homopolymers. The critical concentration required to form a nematic phase in solutions of the block copolymers is correlated with the length (or axial ratio) of the rigid block, and with its proportion in the copolymer.

We report a study of the order parameter, S, for nematic solutions of poly(γ-benzyl-L-glutamate) (PBLG) in dioxane. The homeotropic alignment evolved naturally in thin NaCl cells and was improved by a magnetic field, i.r. spectroscopy was used for determining the ratio of the band intensity in a rather wide composition range and for two different molecular weight PBLG. Bands at 3294 (amide A, N-H stretching) and at 1549 cm−1 (amide II vibration) were used for both isotropic and anisotropic solutions. Angles between the transition moment of the vibrations and the chain axis were selected from the literature. S was found to increase with both polymer concentration and molecular weight, approaching the value of 1 characteristic of perfect orientation. Comparison of the results with the predictions of virial and lattice theories indicates that the former offers the closest representation at rather low polymer volume fraction V2, while the latter is to be preferred at a somewhat larger V2.

A study of the persistence length q and of the critical polymer volume fraction v′2 at which the mesophase appears for poly(n-hexyl isocyanate) (PHIC) in 1-chloronaphthalene is presented. The phase diagram was determined over the temperature range 80 to 188°C, corresponding to v′2 from 0.2 to 1.0. The thermal expansion of the system is accounted for in evaluating v′2 and the axial ratio of the Kuhn segment, 2qd. As reported for PHIC in toluene, and for several cellulose derivatives, the limiting value of v′2 at large molecular weight can be represented rather well by the Kuhn chain model with a temperature-dependent Kuhn segment length. However, while the thermotropic effect is adequately described by the temperature coefficient of q, the experimental v′2 is somewhat smaller than predicted.

The chloroplast-specific elongation factor G (EF-Gchl) of Chlorella vulgaris is detectable only in cells exposed to light for at least 24 h. If chloramphenicol is added to a dark-grown culture, EF-Gchl is not detectable in the cells even after illumination for 24 h. On the contrary, addition of cycloheximide to a dark culture does not reduce significantly the amount of EF-Gchl which is synthesized after the culture has been illuminated for 24 h. Tracer experiments indicate that radioactive amino acids are incorporated into the EF-Gchl of cells treated with cycloheximide. Thus EF-Gchl appears to be synthesized in the chloroplast itself. Some implications of these results are discussed.

The aromatic Polyamide-hydrazide, Monsanto X-500, has been studied by light scattering in dimethyl sulfoxide, a thermodynamically good solvent. The unperturbed dimensions, (〈r〉av/M)1/2 = 1.93 × 10−8 at 25°C., indicate a rather highly extended chain. The persistence length falls in the range 35–63 A, which corresponds to a length of between two and four formula units. This is considerably smaller than the values which have been reported for the aromatic polyamides, poly(p-benzamide) and poly(p-phenylene terephthalamide). X-500 appears to approximate coil-like behavior at a molecular weight of 45,000. Theoretical predictions, based upon the 〈r〉av/bL ratio, are compared with the observation that no evidence for an anisotropic phase has been found in X-500 solutions in dimethyl sulfoxide at polymer volume ranging from 0.12 to 0.19 (depending upon the concentration of added LiCl).

We present new data on the kinetics of band formation during the relaxation of anisotropic solutions of poly(p-benzamide) in dimethyl acetamide + LiCl. The new data, along with previous reports for hydroxypropylcellulose and poly(n-hexylisocianate) solutions, reveal that the band formation time tb decreases with solution viscosity at low shear rates . The occurrence of a new effect is manifested by a minimum in the tb vs dependence. A description in terms of current theoretical concepts is presented.

Equilibre de phase du systeme ternaire polymere rigide, non fractionne (poly p-benzamide)-polymere en pelote, non fractionne (polyacrylonitrile) et un diluant forme de dimethylacetamide et 3% de chlorure de lithium

The phosphorylation reaction of Yamazaki and Higashi was used to prepare wholly aromatic block copolymers containing the rigid units poly(p-benzamide) (PBA) or poly(p-phenylene terephtalamide) (PPD-T) and the flexible units poly(m-phenylene isophthalamide) (MPD-I) or the polyterephthalamide of p-aminobenzhydrazide (PPD-T). Three synthetic procedures were investigated: A) monomers of the flexible block were added to the rigid prepolymer, B) monomers of the flexible block were added to the carboxy terminated rigid prepolymer, and C) the reaction of the carboxy terminated prepolymer of the rigid block with amine terminated prepolymer of the flexible block. Extraction of the copolymer with a non-solvent for the rigid homopolymer indicates that method B) gives the smallest amount of the flexible homopolymer. The extract from the PBA/MPD-I block copolymer was shown to be the flexible MPD-I homopolymer by NMR. The extract from the PBA/PABH-T copolymer could not be characterized by NMR due to overlapping of the spectra, but differential scanning calorimetry was used to identify the extract as PABH-T.

Etude par diffusion de lumiere et viscosimetrie de l'agregation de cellulose dissoute dans DMAC+LiCl

Chloroplasts isolated from spinach leaves carry on a light-driven, RNAase-insensitive incorporation of amino acids. From such chloroplasts an active S-20 fraction was prepared which was capable of incorporating amino acids under the direction of the endogenous mRNA and phenylalanine under that of polyuridylic acid (poly U). Initiation and elongation factors as well as ribosomes active in vitro were obtained from isolated chloroplasts.

A homologous series of polyesters was prepared from 4,4′-dihydroxybiphenyl and dibasic acids having 5-12 methylene units. The mesophases formed at elevated temperatures were studied by differential scanning calorimetry and polarized light microscopy. This family exhibits an unusual odd-even effect when the transition temperatures are plotted as a function of the number of methylene units in the dibasic acid. Not only do the points for odd and even members fall on different curves, but the odd members exhibit a nematic phase over a very short temperature interval, while the even members form a highly ordered smectic phase. For both the odd and even series, the transition temperatures are significantly depressed when the inherent viscosity falls below 0.2 dL/g. The largest depression occurs for the crystal melting transition, so that polymers of low ηinh show anisotropic and biphasic regions over wider temperature ranges. A copolymer formed from an equimolar mixture of sebacic and chiral (+)-3-methyl adipic acid forms a cholesteric phase. Evidently copolymerization destabilizes the smectic phase which would have been expected. The results are discussed in terms of existing theory.