Peptostreptococcus barnesae sp. nov., a Gram-positive,anaerobic, obligately purine utilizing coccus from chicken feces

Anaerobic, Gram-positive cocci were obtained from chicken feces by direct isolation, which grew on the purines uric acid, xanthine, 6,8-dihydroxypurine, guanine, and hypoxanthine. Adenine and glycine were fermented, but not as readily. Acetate, formate, ammonia, and CO₂ were products. The isolated strains were nutritionally non-fastidious, however, they required selenite, molybdate, and tungstate as micronutrients. The cells were spherical and 0.5–0.9 m in diameter. The addition of bile salts enhanced the growth rate in most cases. The organisms proved to be quite resistant to lysis. The guanosine-plus-cytosine (G+C) content of their deoxyribonucleic acid was 33.6 to 34.8 mol%. The peptidoglycan was of the same structure (Gly-Lys-d-Asp) as reported for the anaerobic cocci of Hare group IX. However, the latter strains could only utilize glycine, not purines. Therefore, it is proposed to form a new species, Peptostreptococcus barnesae sp. nov.This paper is dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

Porphyrins in the perienteric fluid of Ascaris lumbricoides.

Cain G. D. and Bassow F. 1976. Porphyrins in the perienteric fluid of Ascaris lumbricoides. International Journal for Parasitology6: 79–82. Porphyrins in the perienteric fluid of adult female A. lumbricoides were esterified in methanolic H₂SO₄, extracted in chloroform, separated by thin-layer chromatography, and identified spectrophotometrically before and after conversion to their zinc and copper chelates. Protoporphyrin IX was the major component, comprising 95·4% of the total; the remaining 4·6 % was coproporphyrin III. Uroporphyrin was not detected; no porphyrins were recovered from other worm tissues. Fluid from worms with light and dark colored guts varied in protoporphyrin content from 0·58 to 4·08 nmoles/ml, respectively, but fluid from both groups contained similar molar ratios of protoporphyrin, coproporphyrin and heme.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX

Carbonic anhydrases (EC 4.2.1.1) catalyse the reversible hydration of CO₂ into bicarbonate and protons. As a hypoxia-sensitive and tumour-associated isoform, isoform CA IX, is significantly overexpressed in various malignancies, being a validated target for new anticancer/antimetastatic drugs. A multitude of studies has shown that CA IX inhibition decreases cancer cell proliferation and metastasis through pHe/pHi modulation and enhancement of ferroptosis among others. Numerous studies demonstrated increased efficacy of cytotoxic drugs combined with CA inhibitors (CAIs) in various cancer types. We tested the inhibitory effect of boric acid (BA), an inorganic Lewis acid, on CA IX as well as other isoforms (CA I, II, and XII). BA acted as a millimolar in vitro CAI, decreased proliferation of two cancer cell lines, although not strong correlations between the in vitro inhibition and in vivo effects were observed. The mechanism of antiproliferative action of BA should be investigated in more detail.     

New coumarin/sulfocoumarin linked phenylacrylamides as selective transmembrane carbonic anhydrase inhibitors: Synthesis and in-vitro biological evaluation

Two novel series of phenylacrylamide linked coumarins and sulfocoumarins (6a-p, 8a-i, and 14a-g) were synthesized and evaluated against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, hCA II, hCA IX and hCA XII for their inhibitory action. All new compounds when screened for carbonic anhydrase inhibitory activity have shown selective inhibition towards the tumor associated isoforms hCA IX and XII over CA I and II, with inhibition constants in the submicromolar to low nanomolar range. Compound 6b and 14g exhibited significant inhibition with low nanomolar potency against hCA IX, whereas 6k was effective against hCA XII. Compounds 6b, 14g and 6k may be considered as lead molecules for future development of cancer therapeutics based on a novel mechanism of action.     

Protoporphyrin IX and oxidative stress

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of δ-amino-levulinic acid synthetase was induced 70–90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2 h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage.

Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.     

Therapeutic angiogenesis for revascularization in peripheral artery disease

Therapeutic angiogenesis for peripheral artery disease (PAD), achieved by gene and cell therapy, has recently raised a great deal of hope for patients who cannot undergo standard revascularizing treatment. Although pre-clinical studies gave very promising data, still clinical trials of gene therapy have not provided satisfactory results. On the other hand, cell therapy approach, despite several limitations, demonstrated more beneficial effects but initial clinical studies must be constantly validated by larger randomized, multi-center, double-blinded, placebo-controlled trials. This review focuses on previous and recent gene and cell therapy studies for limb ischemia, including both experimental and clinical research, and summarizes some important papers published in this field. Moreover, it provides a short comment on combined gene and cell therapy approach on the example of heme oxygenase-1 overexpressing cells with therapeutic properties.     

Discovering novel carbonic anhydrase type IX (CA IX) inhibitors from seven million compounds using virtual screening and in vitro analysis

Carbonic anhydrase type IX (CA IX) enzyme is mostly over expressed in different cancer cell lines and tumor tissues. Potent CA IX inhibitors can be effective for adjusting the pH imbalance in tumor cells. In the present work, we represented the successful application of high throughput virtual screening (HTVS) of large dataset from ZINC database included of ～7 million compounds to discover novel inhibitors of CA IX. HTVS and molecular docking were performed using consequence Glide/standard precision (SP), extra precision (XP) and induced fit docking (IFD) molecular docking protocols. For each compound, docking code calculates a set of low-energy poses and then exhaustively scans the binding pocket of the target with small compounds. Novel CA IX inhibitor candidates were suggested based on molecular modeling studies and a few of them were tested using in vitro analysis. These compounds were determined as good inhibitors against human CA IX target with K_i in the range of 0.85–1.58?μM. In order to predict the pharmaceutical properties of the selected compounds, ADME (absorption, distribution, metabolism and excretion) analysis was also carried out.     

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.