Microbiologic transformation of progesterone by Curvularia clavata Jain

Progesterone was transformed microbiologically by the fungal strain Curvularia clavata Jain. Progesterone (I) was added as substrate when the microorganism reached its exponential growth phase. Three substances were isolated after the fermentation: a non-steroidal substance, radicinin (II), which has been established to be a metabolic product of the fungus and acts as a phytotoxin, and two steroidal substances which resulted from fungal enzymatic action on the progesterone molecule. The structure of each microbial metabolite was elucidated by 1H-nuclear magnetic resonance, mass spectrometry, and infrared and UV analyses, and the yields were determined by high-pressure liquid chromatography. The progesterone metabolites were characterized as 7 alpha,14 alpha-dihydroxypregn-4-ene-3,20-dione (III) and 11 beta, 14 alpha-dihydroxypregn-4-ene-3,20-dione (IV). Evidence for the structure of these steroidal products came from derivatives resulting from acetylation and dehydration.     

Nasitrema attenuata (Trematoda) infection of Tursiops truncatus and its potentialities as an aetiological agent of chronic pulmonary lesions.

Two cases of Nasitrema sp (Trematoda) infection of the bottlenosed dolphin, Tursiops truncatus, are reported and N. attenuata is briefly redescribed. The adults of the trematode occurring in the nasal sinuses and posterior nasal passage of the dolphins are considered as practically harmless for the host but thier eggs, aspirated deep into the bronchial tree, may initiate a foreign-body of inflammatory reaction in the lungs and continuous aspiration of such eggs may provoke a chronic pneumonia condition.     

Isolation and characterization of an aetiological agent in Whipple's disease.

A cell wall deficient form of an alpha-haemolytic streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple''s disease. Serological cross reactivity was shown between the organism and the material within Whipple''s disease macrophages positive for diastase-resistant periodic acid-Schiff (D./P.A.S.). In vitro studies characterized the organism as a facultative intracellular parasite which caused the accumulation within cells of D./P.A.S.-positive material. These results suggest that a pathogenic bacterium is the essential aetiological agent and that the culture of Whipple''s disease tissues in hypertonic media may have practical value.     

Alginate as an antiglycating agent for human serum albumin

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques. The characterization study of AGEs was performed using autofluorescence, fibrillar formation, the increase in absorbance and the quantification of free lysine side chains. The results indicate that alginate inhibits the fructation of HSA as observed by a reduction in the formation of fluorescent AGEs and fibrils. Furthermore, alginate reduces the amount of modified lysine side chains, signified by the lack of increase in absorbance, and increases the helicity of this protein.     

Estradiol as an anti-aromatase agent in human breast cancer cells

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.     

A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the ‘superbug’ Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the ‘Rana Box’, we designed a set of brevinin-1E-OG9 analogues to explore its structure–activity relationship. Brevinin-1E-OG9c-De-NH₂ exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH₂ might represent a promising candidate for the treatment of S. aureus skin infections.     

Identification of a 334-ribonucleotide viral satellite as principal aetiological agent in a tomato necrosis epidemic

Cucumber mosaic virus (CMV), a widespread and economically important virus of vegetable crops, often contains a satellite RNA, here designated CARNA-5 (for CMV-associated RNA 5). Viral satellites are small nucleic acids that are sequence-unrelated to, but replicatively dependent upon, the viral genome. They essentially are molecular parasites of their helper viruses, and thereby frequently modulate viral symptom expression. Some isolates of CARNA-5 change normally moderate CMV symptoms in tomato into a lethal disease named tomato necrosis; others ameliorate CMV symptoms in tomato and other important crop plants. Here we report on the identification and molecular characterization of a 334-nucleotide necrogenic CARNA-5 isolated from tomato fields in southern Italy, where a massive outbreak of lethal necrosis occurred in the summer of 1988. This is the first time that direct evidence is given for the involvement of a viral satellite in a crop disease of epidemic scale. The possible molecular interrelationships between plant, virus, satellite and other factors that influence the satellite-induced symptom modulation underlying such a catastrophe are discussed.     

Preparation of squid skin collagen hydrolysate as an antihyaluronidase,antityrosinase, and antioxidant agent

A collagen was isolated from squid skin, a processing waste product. The biofunctional activities of enzymatic squid skin collagen hydrolysates were determined to produce a value-added material. Five low-molecular-mass hydrolysate fractions, F1 (>30 kD), F2 (10–30 kD), F3 (3–10 kD), F4 (1–3 kD), and F5 (<1 kD), were manufactured from its enzymatic hydrolysate by ultrafiltration. Fraction F3 had the strongest antihyaluronidase inhibitory activity. Gly, Val, and Pro were major amino acids in F3, while Met, Tyr, and His were minor ones. The molecular mass of F3 was in the range of 3.4 to 10 kD. F3 exhibited copper chelating ability in a concentration-dependent manner. The ferrous chelating ability of F3 was almost 50% at 200 µg/mL. F3 also inhibited tyrosinase activity by 39.65% at 1 mg/mL. Furthermore, F3 had stronger hydroxyl radical scavenging activity (IC₅₀ = 149.94 µg/mL) than ascorbic acid (IC₅₀ = 212.94 µg/mL). Therefore, the squid collagen hydrolysate can be utilized as a nutraceutical or cosmeceutical agent.     

Thalidomide as an anti-cancer agent

Thalidomide is a glutamic acid derivative initially introduced as a sedative hypnotic nearly forty years ago. It was withdrawn following numerous reports linking it to a characteristic pattern of congenital abnormalities in babies born to mothers who used the drug for morning sickness. It has gradually been re-introduced into clinical practice over the past two decades, albeit under strict regulation, since it was found to be useful in the management of erythema nodosum leprosum and HIV wasting syndrome. Recognition of its anti-angiogenic effect led to its evaluation in the treatment of various malignancies, where angiogenesis has been shown to play an important role. Numerous clinical trials done over the past four years have confirmed the significant anti-myeloma activity of this drug. It has also shown promise in preliminary trials in the treatment of a variety of different malignant diseases. The mechanisms of its antineoplastic effects continue to be the focus of ongoing research. It has become clear that even though its anti angiogenic effects play a significant role in the anti-tumor activity, there are other properties of this drug which are responsible as well. It also possesses anti-TNF alpha activity, which has led to its evaluation in several inflammatory states. In this concise review, we briefly describe the historical background and pharmacological aspects of this drug. We have concisely reviewed the current knowledge regarding mechanisms of its anti-neoplastic activity and the results of various clinical trials in oncology.     

Reserpine as an uncoupling agent

1. Reserpine, like the uncoupling agent, 2,4-dinitrophenol prevents oxidative phosphorylation but stimulates the rate at which oxygen is reduced.

2. Both reserpine and 2,4-dinitrophenol fail to stimulate oxygen uptake by isolated mitochondria in the presence of arginine.

3. Both 2,4-dinitrophenol and reserpine induce proton permeability in the mitochondrial membrane so that H⁺ is absorbed from the suspending medium.

4. When the reaction system contains reserpine, accumulation of Ca²⁺ by mitochondria is inhibited.

5. Reserpine decreases both ADP:O and P:O ratios which strongly suggest that reserpine is an uncoupling agent.     

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.     

Methylene blue as an antimalarial agent

Abstract

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

Drosophila cecropin as an antifungal agent

The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 microM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture.     

Phytoestrogen genistein as an anti-staphylococcal agent

The soybean-derived isoflavone genistein has been shown to exert beneficial effects on many disorders, including cancer and cardiovascular diseases. The effects of genistein on mammalian cells are mediated by its abilities to inhibit topoisomerase II and protein tyrosine kinase. In order to examine the potential antibacterial activities of genistein, we incubated the bacteria with various concentrations of this compound for different periods of time and assessed the viable counts. Exposure to genistein exhibited an inhibitory effect on all staphylococcal strains tested, including methicillin-resistant strains. Furthermore, the growth of Streptococcus pasteurianus, Bacillus cereus, and Helicobacter pylori was clearly inhibited by genistein, whereas Escherichia coli growth was not suppressed. Daidzein, which is structurally similar to genistein, but deficient in topoisomerase II inhibitory activity, also inhibited the growth of Staphylococcus aureus, albeit with lower potency than genistein. Our results indicate that genistein exerts potent antibacterial properties in vitro, which are possibly mediated by the stabilization of the covalent topoisomerase II-DNA cleavage complex.     

Methylene blue as an antimalarial agent

Methylene blue has intrinsic antimalarial activity and it can act as a chloroquine sensitizer. In addition, methylene blue must be considered for preventing methemoglobinemia, a serious complication of malarial anemia. As an antiparasitic agent, methylene blue is pleiotropic: it interferes with hemoglobin and heme metabolism in digestive organelles, and it is a selective inhibitor of Plasmodium falciparum glutathione reductase. The latter effect results in glutathione depletion which sensitizes the parasite for chloroquine action. At the Centre de Recherche en Santé de Nouna in Burkina Faso, we study the combination of chloroquine with methylene blue (BlueCQ) as a possible medication for malaria in endemic regions. A pilot study with glucose-6-phosphate dehydrogenase-sufficient adult patients has been conducted recently.     

Agrobacterium tumefaciens as an agent of disease

Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer. Since this discovery, A. tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange. It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology. These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research. Now, the diverse areas of A. tumefaciens research are again converging because new discoveries in transformation biology and the use of A. tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.     

Soluble IL-2 receptor as an agent of serum-mediated suppression in human visceral leishmaniasis

In visceral leishmaniasis (VL), patient's lymphocytes are not activated by leishmania Ag stimulation, and their sera exhibit a potent nonspecific suppressive effect on the responses of normal lymphocytes. Sera were obtained from 33 VL patients, eight patients with subclinical VL, and from 27 normal volunteers. Only sera from VL patients markedly reduced Con A-induced lymphocyte proliferative responses, as well as IL-2 or IFN-gamma production by normal lymphocytes. Addition of exogenous human rIL-2 to cultures containing VL patient sera partially reversed the normal lymphocyte proliferative capacity and restored IFN-gamma production. This phenomenon was consistent with the presence of greatly elevated levels of soluble IL-2R (sIL-2R) in VL patients' sera (4299 +/- 2351 U/ml), well above those of normal sera (180 +/- 94 U/ml), or of sera from patients with subclinical leishmania infection without immunosuppression (1002 +/- 281 U/ml). Furthermore, the removal of sIL-2R reduced VL serum suppressive activity as evaluated by effects on IL-2 and on IFN-gamma production. These data suggest the participation of high levels of sIL-2R in the serum-mediated suppression in VL.