In vitro acitivity of ribostamycin against Prototheca sp.

The in vitro activity of ribostamycin against algae of the genus Prototheca was evaluated by minimum inhibitory concentration (MIC) studies on solid media. Concentrations of 4 mcg/ml were required to inhibit 100% of the P. zopfii strains; 16 mcg/ml inhibited 100% of the P. stagnora strains and 95% of the P. wickerhamii strains. These values are inferior to plasma concentrations obtained after injection of ribostamycin. It is likely that this antibiotic could be effective in the treatment of protothecosis in man.     

Identification of the Prototheca Species by Immunofluorescence

Studies were carried out to develop fluorescent antibody reagents for the identification of the Prototheca species and for their differentiation from morphologically similar fungi of various genera in formalin-fixed tissues. Antisera against representative isolates of P. filamenta, P. moriformis, P. stagnora, P. wickerhamii, and P. zopfii were produced in rabbits. Antiglobulins, labeled with fluorescein-isothiocyanate that intensely stained most cells of the homologous species, were selected for use as potential diagnostic reagents. By adsorbing the conjugates with selected heterologous cross-staining protothecae, reagents that were both sensitive and specific were obtained. Evaluation of the adsorbed conjugates with sections of tissue infected with protothecae, sections of tissue infected with morphologically similar fungi, and cultures of protothecae showed that these reagents are useful for the rapid and reliable identification of the Prototheca species.     

Appearance of Colonies of Prototheca on CHROMagar Candida medium

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum. This revised version was published online in August 2006 with corrections to the Cover Date.     

A technique for isolating pure, viable parenchymal cells and their nuclei from rat mammary gland

Parenchymal cell preparations of high purity and of sufficient yield for biochemical studies were obtained by treatment of rat mammary gland tissue with a curde preparation of collagenase and trypsin in tissue culture medium 199, followed by filtration. The isolated cells, obtained in approximately 2 hr, were of acceptable quality as determined by (a) their appearance in the light microscope, (b) their ability to exclude trypan blue, and (c) their ability to incorporate [³H]lysine into cellular protein in vitro. A method is also described for purifying parenchymal cell nuclei in high yield by ultracentrifugation in dense sucrose. The isolated nuclei were of acceptable quality as judged by (a) their appearance in the light microscope and (b) their RNA, DNA, and protein content.     

Pathogen-induced plant proteins

Pathogen-induced plant proteins are classified by their functional characteristics: (a) involvement in plant cell signaling; (b) inhibition of enzymes excreted by the pathogens; (c) stabilization of plant cell walls or ability to trigger apoptosis; (d) enzymatic activity producing lysis of cell walls of pathogenic fungi and bacteria; (e) enzymatic activity in metabolic pathways of phenylpropane and terpene phytoalexins; and (f) ability to affect the pathogens directly, by disturbing the function of their cell membranes or by inactivating their ribosomes. Examples of transgenic plants with increased immunity against pathogens are also provided.     

Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula

The physiological mechanisms controlling plant responses to dynamic changes in ambient solar ultraviolet (UV) radiation are not fully understood: this information is important to further comprehend plant adaptation to their natural habitats. We used the fluorimeter Dualex to estimate in vivo the epidermal flavonoid contents by measuring epidermal UV absorbance (A(375) ) in Betula pendula Roth (silver birch) leaves of different ages under altered UV. Seedlings were grown in a greenhouse for 15 days without UV and transferred outdoors under three UV treatments (UV-0, UV-A and UV-A+B) created by three types of plastic film. After 7 and 13 days, Dualex measurements were taken at adaxial and abaxial epidermis of the first three leaves (L1, L2 and L3) of the seedlings. After 14 days, some of the seedlings were reciprocally swapped amongst the treatments to study the accumulation of epidermal flavonoids in the youngest unfolded leaves (L3) during leaf expansion under changing solar UV environments. A(375) of the leaves responded differently to the UV treatment depending on their position. UV-B increased the A(375) in the leaves independently of leaf position. L3 quickly adjusted A(375) in their epidermis according to the UV they received and these adjustments were affected by previous UV exposure. The initial absence of UV-A+B or UV-A, followed by exposure to UV-A+B, particularly enhanced leaf A(375) . Silver birch leaves modulate their protective pigments in response to changes in the UV environment during their expansion, and their previous UV exposure history affects the epidermal-absorbance achieved during later UV exposure.     

Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or CO2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5'-triphosphates or deoxyribonucleoside 5'-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, and dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.     

Sensitivity analysis of the tree distribution model Phenofit to climatic input characteristics: implications for climate impact assessment

Species distributions are already affected by climate change. Forecasting their long‐term evolution requires models with thoroughly assessed validation. Our aim here is to demonstrate that the sensitivity of such models to climate input characteristics may complicate their validation and introduce uncertainties in their predictions. In this study, we conducted a sensitivity analysis of a process‐based tree distribution model Phenofit to climate input characteristics. This analysis was conducted for two North American trees which differ greatly in their distribution and eight different types of climate input for the historic period which differ in their spatial (local or gridded data) and temporal (daily vs. monthly) resolution as well as their type (locally recorded, extrapolated or simulated by General Circulation Models). We show that the climate data resolution (spatial and temporal) and their type, highly affect the model predictions. The sensitivity analysis also revealed, the importance, for global climate change impact assessment, of (i) the daily variability of temperatures in modeling the biological processes shaping species distribution, (ii) climate data at high latitudes and elevations and (iii) climate data with high spatial resolution.     

Isolation and characterisation of a non-polyadenylated mRNA species with an affinity for poly(adenylic acid) from Friend leukaemia cells

Utilizing the technique of poly(A)-Sepharose affinity chromatography, it is possible to isolate a novel class of RNA molecules from polysomes of Friend leukaemia cells. These RNA species display messenger RNA-like behaviour. They are released from polysomes on treatment with EDTA and are able to direct polypeptide synthesis in a cell-free protein synthesising system. They appear to be distinct from the polyadenylated mRNAs, as judged by their lack of a 3'-terminal poly(A) tract, by their different size distribution, by their unusual base composition, by the presence of a possible 'uridylate rich' region towards their 3'-end, by their low sequence homology to polyadenylated mRNAs and by the difference in at least some of their translation products.     

Antibacterial dimeric copper(II) complexes with chromone-derived compounds

A new series of six chromone-derived compounds and their Cu(II) complexes have been synthesized and characterized by their physical, spectral and analytical data. The ligands and their Cu(II) complexes were screened for their in vitro antibacterial activity against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri) and two Gram-positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains by agar-well diffusion method. The ligands were found to exhibit either no or low-to-moderate activities against one or more bacterial species whereas, the Cu(II) complexes exhibited moderate-to-high activity. The ligands which were inactive before complexation became active upon complexation with the Cu(II) metal ion and less active became more active.     

Observations of the primordial germ cells in blood samples from the chick embryo

Primoridal germ cells (PGCs) from the circulating blood in 2-day chick embryos were observed by histochemical techniques in smear preparation and by phase contrast microscopy in fresh samples. In the blood smear, PGCs were readily distinguished from blood cells by their large size (15–20 μm in diameter) and by the round and large nuclei (8–10 μm in diameter) occupying eccentrically the greater parts of the cells. Histochemically, they were demonstrated to contain abundant glycogen, a lesser amount of yolk granules (by the PAS reaction), and a large amount of lipid droplets (by the Oil red O and Sudan black B stains) in their cytoplasm. Alkaline phosphatase activity was proved slightly in their cytoplasm by the simultaneous diazo coupling method. In phase contrast microscopy, living PGCs in the blood and from the primitive gonad of 3-day embryos in parts of the examinations were observed to change their shape in somewhat amoeboid fashion, suggesting their capability for locomotion as a possible mechanism of the migration of PGCs in vivo.     

Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion

When added to cultures of parathyroid hormone (PTH)- bones, dichloromethylenebisphosphonate (C12MBP) and 3-amino-1-hydroxypropydilene-1,1-bisphosphonate (AHPrBP) inhibit completely and in a parallel manner the development of resorption lacunae, the loss of calcium by the explants and their PTH-induced excretion of lysosomal hydrolases (β-glucuronidase and N-acetyl-β-glucosaminidase). The loss of collagen (hydroxyproline) by the bones is usually less inhibited than their loss of calcium and their heparin-induced excretion of collagenase is unaffected. To interpret these data, it is proposed that these bisphosphonates act more on the activity of osteoclasts, suppressing simultaneously their excretion of lysosomal enzymes and their erosion of mineralized bone matrix, than on that of other cell types (osteoblasts ?) responsible for collagenase production and the removal of uncalcified collagen.     

Studies of fluorescence depolarization at high pressures of dimyristoylphosphatidylcholine liposomes containing poly(gamma-benzyl-L-glutamate)

The behaviors of four kinds of poly(gamma-benzyl-L-glutamate) (PBLG) molecules and their locations in dimyristoyl-phophatidylcholine (DMPC) liposomes were studied by fluorescence techniques at high pressures of up to 981 bar. The fluorescent substances 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-aminonaphthalene-8-sulfonate (ANS) and PBLGs labeled with a dansyl group were used as probes of hydrophobic and hydrophilic spheres and the motions of polypeptides, respectively. The changes in mobilities of the PBLG molecules depended on their concentrations, degrees of polymerization and the lengths of hydrocarbon chains attached to their terminals. The molecular motions of the PBLGs were altered by increase in their contents, caused by squeezing the PBLG molecules out of the membranes. Models of the membranes and the behavior of polypeptides in the membranes at high pressures are discussed.     

Biliary bile acids in birds of the Cotingidae family: taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid

Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5β-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.     

Mutual recognition of sphingolipid molecular species in membranes

In membranes liquid disordered (l(d)) and liquid ordered (l(o)) domains can exist that differ in fluidity and function. L(o) areas are predominantly composed of cholesterol and sphingomyelin (SM). Study of the formation of such domains is hampered by a lack of methods to analyze specific lipid-lipid interactions at low concentrations of individual molecular lipid species in membranes. Here, we developed a simple biophysical method to experimentally assess the affinity of various molecular species of SM for cholesterol, and for their endogenous counterparts (kin) at physiological concentrations. Fluorescent SM (flc SM) molecular species with a conjugated pentaene system in their fatty acids are employed to monitor their affinity to either cholesterol or their kin by fluorescence unquenching. With this novel method we show that specific interactions of individual SMs with cholesterol or their kin exist, indicating the presence of SM nano-domains in l(d)-phases, strictly based on kin/cholesterol recognition.     

Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease

Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A.     

Food partitioning between breeding White-tailed Kites (Elanus leucurus; Aves; Accipitridae) and Barn Owls (Tyto alba; Aves; Tytonidae) in southern Brazil.

I examined the diet of breeding White-tailed Kites (Elanus leucurus; Aves; Accipitridae) and Barn Owls (Tyto alba; Aves; Tytonidae) in an agrarian area of southern Brazil by analyzing regurgitated prey remains. The objective was to evaluate how these raptors, which differ markedly in their hunting activity periods (owls are nocturnal and kites diurnal), share their mammalian food component. 2,087 prey consumed by Barn Owls and 1,276 by White-tailed Kites were identified. They presented a high overlap of food-niches (Piankas index was 0.98). Based on the daily activity period of their main small mammal prey, a lower overlap would be expected. The crepuscular/nocturnal Mus musculus was the main prey for the diet of breeding Barn Owls (81%) and White-tailed Kites (63%). This small exotic rodent provided 63% of the small mammal biomass ingested by owls and 44% by kites. Larger native small mammals were also considered important for the diet of kites, mainly because of their biomass contribution. Although these raptors differ markedly in their hunting activity periods, Barn Owls and White-tailed Kites are very similar predators in southern Brazil, overlapping their diets.     

Ultrastructure of the meningococcus in logarithmic and stationary development phases

The complex study of Neisseria meningitidis cultures A-208 in the time course of their development has disclosed that broth cultures in the logarithmic and stationary phases of their development are most valid on account of all their biological properties (the specific character of the reaction of agglutination, viability, the morphology of colonies and cells in light and electron microscopy). The use of scanning electron microscopy has made it possible to reveal bubbly endotoxin excretion in N. meningitidis and funnel-shaped depressions on their surface corresponding, probably, to nucleoid epicenters . In ultrathin sections some previously unknown features of the ultrastructure of N. meningitidis in the logarithmic and stationary phase of their development have been detected: (a) the morphological heterogeneity of N. meningitidis represented by cells of the "light" (L) and "dark" (D) types; (b) the surface structures of meningococcal cells from the cultures in the stationary phase of development show the tendency to smoothing out, which is accompanied by their sharper differentiation.     

Nitrate transporters in plants: structure, function and regulation

Physiological studies have established that plants acquire their NO(-3) from the soil through the combined activities of a set of high- and low-affinity NO(-3) transport systems, with the influx of NO(-3) being driven by the H(+) gradient across the plasma membrane. Some of these NO(-3) transport systems are constitutively expressed, while others are NO(-3)-inducible and subject to negative feedback regulation by the products of NO(-3) assimilation. Here we review recent progress in the characterisation of the two families of NO(-3) transporters that have so far been identified in plants, their structure and their regulation, and consider the evidence for their roles in NO(-3) acquisition. We also discuss what is currently known about the genetic basis of NO(-3) induction and feedback repression of the NO(-3) transport and assimilatory pathway in higher plants.