Javanicin, an antibacterial naphthaquinone from an endophytic fungus of neem, Chloridium sp.

The endophytic fungus Chloridium sp. produces javanicin under liquid and solid media culture conditions. This highly functionalized naphthaquinone exhibits strong antibacterial activity against Pseudomonas spp., representing pathogens to both humans and plants. The compound was crystallized and the structure was elucidated by X-ray crystallography. The X-ray structure confirms the previously elucidated structure of the compound that was done under standard spectroscopic methods. The importance of javanicin in establishing symbiosis between Chloridium sp. and its host plant, Azadirachta indica, is briefly discussed.

Colutellin A, an immunosuppressive peptide from Colletotrichum dematium.

Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 microg ml(-1) (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and beta-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4(+) T-cell activation of interleukin 2 (IL-2) production with an IC(50) of 167.3+/-0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 microg ml(-1) exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.

Purification, identification and activity of phomodione, a furandione from an endophytic Phoma species.

Phomodione, [(4aS(*),9bR(*))-2,6-diacetyl-7-hydroxy-4a,9-dimethoxy-8,9b-dimethyl-4a.9b-dihydrodibenzo[b,d]furan-1,3(2H,4H)-dione], an usnic acid derivative, was isolated from culture broth of a Phoma species, discovered as an endophyte on a Guinea plant (Saurauia scaberrinae). It was identified using NMR, X-ray crystallography, high resolution mass spectrometry, as well as infrared and Raman spectroscopy. In addition to phomodione, usnic acid and cercosporamide, known compounds with antibiotic activity, were also found in the culture medium. Phomodione exhibited a minimum inhibitory concentration of 1.6 microg/mL against Staphylococcus aureus using the disk diffusion assay, and was active against a representative oomycete, ascomycete and basidiomycete at between three and eight micro-grams per mL.

Muscodor albus E-6, an endophyte of Guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant.

Muscodor albus is an endophytic fungus, represented by a number of isolates from tropical tree and vine species in several of the world's rainforests, that produces volatile organic compounds (VOCs) with antibiotic activity. A new isolate, E-6, of this organism, with unusual biochemical and biological properties, has been obtained from the branches of a mature Guazuma ulmifolia (Sterculiaceae) tree growing in a dry tropical forest in SW Ecuador. This unique organism produces many VOCs not previously observed in other M. albus isolates, including butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, N-(1-methylpropyl); and certain azulene and naphthalene derivatives. Some compounds usually seen in other M. albus isolates also appeared in the VOCs of isolate E-6, including caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl- derivatives. The biological activity of the VOCs of E-6 appears different from the original isolate of this fungus, CZ-620, since a Gram-positive bacterium was killed, and Sclerotinia sclerotiorum and Rhizoctonia solani were not. Scanning electron micrographs of the mycelium of isolate E-6 showed substantial intertwining of the hyphal strands. These strands seemed to be held together by an extracellular matrix accounting for the strong mat-like nature of the mycelium, which easily lifts off the agar surface upon transfer, unlike any other isolate of this fungus. The ITS-5.8S rDNA partial sequence data showed 99 % similarity to the original M. albus strain CZ-620. For the first time, successful establishment of M. albus into its natural host, followed by recovery of the fungus, was accomplished in seedlings of G. ulmifolia. Overall, isolates of M. albus, including E-6, have chemical, biological and structural characteristics that make them potentially useful in medicine, agricultural and industrial applications.

Liver transplantation at the Cuban Center for Medical and Surgical Research.

From July 4, 1999, when a liver transplantation program was started in Cuba, to October 2003, 66 procedures had been performed in 60 patients. The most frequent reason was cirrhosis caused by hepatitis C virus (29%), and alcoholic cirrhosis (22%). Two patients received simultaneous liver-kidney transplants. Half of the patients were men. Patient ages ranged from 12 to 62 years; the average surgical time was 6 hours; and cold ischemia time was 4 to 14 hours. The average blood consumption was 2033 mL; 2900 mL of plasma and 8 units of platelets were used in 7 cases. Immunosuppression was mainly cyclosporine (Neoral), mycophenolate mofetil or azathioprine, and prednisone. Acute cellular rejections were treated in almost all cases with 3 doses of methylprednisolone. The most frequent complications were biliary (24%), hepatic arterial thrombosis (12%), post-surgical bleeding (10%), acute cellular rejection (24%), and ductopenic rejection (2%). The overall 1-year survival rate was 73.7%.

Coronamycins, peptide antibiotics produced by a verticillate Streptomyces sp. (MSU-2110) endophytic on Monstera sp.

Coronamycin is a complex of novel peptide antibiotics with activity against pythiaceous fungi and the human fungal pathogen Cryptococcus neoformans. It is also active against the malarial parasite, Plasmodium falciparum, with an IC(50) of 9.0 ng ml(-1). Coronamycin is produced by a verticillate Streptomyces sp. isolated as an endophyte from an epiphytic vine, Monstera sp., found in the Manu region of the upper Amazon of Peru. Bioassay-guided fractionation of the fermentation broths of this endophyte on silica gel and HPLC chromatography yielded two principal, inseparable, peptides with masses of 1217.9 and 1203.8 Da. Three other minor, but related components, are also present in the preparation. Amino acid analysis of coronamycin revealed residues of component 1, component 2, methionine, tyrosine and leucine at a ratio of 2:2:1:1:3. Other compounds with antifungal activities are also produced by this endophytic streptomycete.

The transplantation donation process in the Centro de Investigaciones Medico Quirurgicas of Cuba: 1999-2002.

OBJECTIVE: In 1998 in the Centro de Investigaciones Medico Quirurgicas the Transplant Coordination Office (TCO) was created, with the aim to organize a system to support a hepatic transplantation program. This organization, which changed the transplantation-donation process not only in our center but in the whole country, is described in this article. METHOD: The files of donors generated in our hospital were studied together with the transplant coordination records, from 1999 till the first half of 2002. RESULTS: In the period studied, 21 potential donors were diagnosed with brain death, yielding a donation rate of 71.4%. Brain death was most frequently caused by vascular brain disease; however, in the realized donor group, the cranioencephalic trauma predominated. The typical donor was a man of average age 39.2 years (range, 18-86 years). Among the potential donors, 24% were excluded based on medical criteria, and 5% due to family objections. Forty liver transplantation were performed in 36 patients including 1 liver-kidney simultaneous procedure. The principal etiologies for transplant included hepatitis C virus cirrhosis, 22%; alcoholic, 19%; and acute hepatic failure, 13%. Kidney transplantations were performed in 70 patients, including 41 from cadaveric donors (53.6%) and 29 from living related donors (41.4%). In 2001, a pancreas-kidney transplantation program was started. CONCLUSION: The creation of the TCO has been of paramount importance to optimize transplantation program functions.

Kakadumycins, novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia.

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.

Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans.

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.

Evidence for purinergic neurotransmission in the urinary bladder of pithed rats.

The purpose of this study was to investigate the contribution of adenosine-5'-triphosphate (ATP) to segmental (L6-S2) spinal electrical stimulation evoked increases in intra-vesical pressure in pithed rats. Exogenous ATP and substance P produced dose-dependent increases in intra-vesical pressure (ED10 mmHg (dose required to elicit 10 mmHg increase in intra-vesical pressure)= 1.7 mg/kg and 1.1 microg/kg, i.v., respectively). Desensitisation (or antagonism) of P2x purinoceptors with alpha,beta-methylene ATP (alpha,beta-meATP; 30 microg/kg per min, i.v.) or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 mg/kg, i.v.) significantly (p < 0.05) antagonized the intra-vesical pressure responses to ATP (> 8 and 3.6-fold increase in ED10 mmHg, respectively) but had no significant effect on intra-vesical pressure responses to substance P. Spinal stimulation evoked frequency-dependent increases in intra-vesical pressure (EF20 mmHg (frequency required to produce 20 mmHg increase in intra-vesical pressure) = 3.4 Hz). Blockade of muscarinic cholinoceptors and adrenoceptors with atropine (3 mg/kg, i.v.), propranolol (3 mg/kg, i.v.) and phentolamine (10 mg/kg, i.v.) produced marginal attenuation of the intra-vesical pressure responses to spinal stimulation indicating a major non-adrenergic non-cholinergic (NANC) component in the overall response. The NANC responses were significantly (p < 0.05) antagonized by alpha,beta-meATP (30 microg/kg per min, i.v.) and PPADS (10 mg/kg, i.v.) (> 2.6-fold increase in EF20 mmHg), consistent with involvement of a purinergic neurotransmitter, presumably ATP. Comparative studies in young (4-6 months) and old (21-23 months) Fischer rats revealed no age-dependent changes in the relative contribution of the cholinergic and purinergic systems, with the latter being the dominant one. These findings suggest that purinergic neurotransmission, presumably mediated by ATP acting via P2x purinoceptors, represents a major component of excitatory innervation to the urinary bladder in pithed rats.

Dramatic response of follicular thyroid carcinoma with superior vena cava syndrome and tracheal obstruction to external-beam radiotherapy.

We report a patient with follicular thyroid carcinoma progressing to superior vena cava (SVC) syndrome and tracheal obstruction despite multiple doses of radioactive iodine therapy but subsequently responding dramatically to external-beam radiotherapy (RT). Although RT is not considered to be the treatment of choice for follicular carcinoma, RT in our patient produced unequivocal improvement of SVC syndrome and tracheal obstruction.

Cytoplasmic retinoid-binding proteins and retinoid effects on insulin release in RINm5F beta-cells.

Vitamin A (retinol) is required for insulin secretion, and retinoic acid substitutes for retinol in this function. To determine if retinol acts at the beta-cell level, we assayed beta-cells of the rat insulinoma (RINm5F) line for cytosolic retinol- and retinoic acid-binding proteins (CRBP and CRABP) by radioimmunoassay (RIA) and [3H]retinol and [3H]retinoic acid binding to cytosol extracts. Furthermore, we tested whether insulin release from cells was affected by addition of retinol or retinoic acid to culture medium. RINm5F cells were grown to near confluence before assay of CRBP and CRABP. Scatchard analysis showed the Kd for retinol to be approximately 6 nM at a level of 4.5 pmol/mg protein or 300,000 sites/cell. Sucrose density-gradient assay showed single discrete peaks migrating at 2S for both retinol and retinoic acid. RIA of whole-cell extracts showed CRBP and CRABP levels of 5.27 +/- 0.41 and 2.95 +/- 0.75 pmol/mg protein, respectively. Retinol (1.75 microM) and retinoic acid (0.175 and 1.75 microM) increased KCl-induced insulin release. Considered together, the presence of CRBP and CRABP in a beta-cell line and the increase in KCl-induced insulin release by retinol and retinoic acid are consistent with the idea that retinol has a functional role in insulin secretion and suggest a potential mechanism of action at the beta-cell level similar to that observed in other retinoid-responsive cells.