Nostoc muscorum: a regioselective biocatalyst for 17-carbonyl reduction of androst-4-en-3,17-dione and androst-1,4-dien-3,17-dione

Nostoc muscorum PTCC 1636 was examined for its ability to convert androst-4-en-3,17-dione (AD) and androst-1,4-dien-3,17-dione (ADD) to their 17-hydroxy related derivatives in BG-11 medium. Bioconversion procedures were carried out at 25 °C without shaking. The metabolites obtained were purified using chromatographic methods and characterized as testosterone and 1-dehydrotestosterone on the basis of their spectroscopic features. In both cases, the bioreaction characteristics observed were 17-carbonyl reduction.     

Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.     

Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum

Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was, isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11beta-hydroxytestosterone, 11beta-hydroxyandrost-4-en-3,17-dione and 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.     

Fluorescent quantification of size and lamellarity of membrane nanotubes

Membrane nanotubes, ubiquitous in cellular systems, adopt a spectrum of curvatures and shapes that are dictated by their intrinsic physical characteristics as well as their interactions with the local cellular environment. A high bending flexibility is needed in the crowded cytoplasm where tubes often need to bend significantly in the axial direction at sub-micron length scales. We find the stiffness of spontaneously formed membrane nanotubes by measuring the persistence length of reconstituted membrane nanotubes freely suspended in solution and imaged by fluorescence microscopy. By quantifying the tube diameter we demonstrate for the first time that the persistence length scales linearly with radius. Although most tubes are uni-lamellar, the predicted linear scaling between tube radius and persistence length allows us to identify tubes that spontaneously form as multilamellar structures upon hydration. We provide the first experimental evidence that illumination of lipid fluorophores can have a profound effect on the lipid bilayer which we sensitively detect as a continuous change in the tube persistence length with time. The novel assay and methodology here presented has potential for quantification of the structural reinforcement of membrane tubes by scaffolding proteins.     

Novel cyclized Pifithrin-alpha p53 inactivators: synthesis and biological studies

Starting from various cyclic or bicyclic ketones, we have synthesized novel Pifithrin-alpha analogues bearing different methyl substituted phenyl ketone groups at the N3-position of the 2-iminothiazole heterocycle. From stability studies in a biological medium as well as under specific chemical conditions, we have shown by NMR techniques that through a dehydration process, some derivatives can generate their corresponding cyclized analogues. All of the new analogues, Pifithrin-like and polycyclic dehydrated derivatives were assessed for their p53 inactivation potency by measuring survival of cortical neurons, whose death was induced by the DNA-damaging agent etoposide. Pifithrin-alpha like 2f as well as the cyclic dehydrated 6b analogue were found to be one log more potent p53 inactivators than reference compound Pft-alpha, with EC50 values ranging around 30 nM. These results support the finding that p53 inactivation by Pft-alpha analogues could be also due to the presence of the cyclic dehydrated Pft-alpha forms, generated in situ in the biological assay incubation medium.     

Functional analysis of cells obtained from bronchoalveolar lavage fluid (BALF) of lung cancer patients

BALF from tumor segments provides access to immune system cells in contact with lung tumors. We analyzed BALF cells as to their production of H2O2 and NO, comparing tumor-affected to non-affected lung segments. Twelve patients were studied (4 NSCLC, 3 SCC, 5 Adenocarcinoma). The cell numbers recovered from BALF varied, and, in adenocarcinoma patients, smaller numbers were recovered from tumor-affected segments. H2O2 production (up to 6.3 nmoles/2x10(5)cells) was obtained in 7/12 patients and, in these, it was more frequent in non-affected segments (7/7) than in affected segments (2/7). After culture, NO production was observed in three patients (6 to 314 microM) that also produced H2O2. These functional characteristics of cells in contact with neoplasia may have a role in determining the fate of the interactions between the immune system and lung cancer.     

Synthesis and characterization of a photo-cross-linked biodegradable elastomer

The synthesis and characterization of a photocurable biodegradable elastomer as a potential biomaterial for the delivery of thermosensitive drugs are described. The elastomer was prepared from UV initiated cross-linking of an acrylated star-poly(epsilon-caprolactone-co-D,L-lactide) prepolymer. The influence of the molecular weight of the acrylated prepolymer on the final elastomer mechanical and thermal properties was determined. The glass-transition temperature of the elastomers was independent of the prepolymer molecular weight and was from -6 to -8 degrees C. The Young's modulus and stress at break of the elastomers was proportional to the inverse of the prepolymer molecular weight, while the strain at break increased in a linear fashion with the prepolymer molecular weight. Over a degradation period of 12 weeks in phosphate buffered saline, the elastomers exhibited little mass loss, appreciable mechanical strength loss, and little dimensional or strain at break change.     

A genome annotation-driven approach to cloning the human ORFeome

We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.     

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.