Enhancement of stability of biosurfactant produced by <Emphasis Type="Italic">Candida lipolytica</Emphasis> using industrial residue as substrate

This work describes experimental results carried out on the fermentation of Candida lipolytica, which produced a new biosurfactant when grown on a vegetable oil refinery residue as substrate. The cell-free culture broth containing the biosurfactant formed stable emulsions with hydrophobic natural compounds. Emulsification properties of the biosurfactant were not affected by salinity; however, treatment at a higher temperature decreased the emulsification activity, indicating applications in oil recovery. The isolated biosurfactant corresponds to a yield of 4.5 g/l, and the surface tension of water was reduced from 71 to 32 mN/m. Preliminary chemical characterizations showed that the biosurfactant consisted of protein (50%), lipid (20%), and carbohydrate (8%).     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C₂/C₁₈ reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.     

Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine, parasitic nematode, Ascaris suum

The acidic glycolipid fraction (AF) of the porcine, parasitic nematode, Ascaris suum , consisted of two subfractions. The major component AF II reacted with orcinol-sulfuric acid and molybdate, while the minor component AF I gave a positive reaction with azure-A, a cationic dye specific for sulfatides. Sugar constituent analysis, methanolysis, methylation analysis, matrix-assisted laser desorption/ionization time- of-flight mass spectrometry, liquid secondary-ion mass spectrometry, and gas-liquid chromatography/mass spectrometry specified AF II to be an unusual phosphoinositolglycosphingolipid (Galalpha1-Ins-P-1ceramide) and the minor component AF I to be a 3-sulfogalactosylcerebroside (HSO3- 3Galss1-1ceramide). The ceramide moiety of both components consisted of lignoceric (C24:0) and cerebronic (C24h:0) acids and mainly C17 iso- branched sphingosine. Immunohistochemical localization studies of the glycolipid-bound antigenic determinants with a polyclonal antiserum against AF II and an anti-sulfatide monoclonal antibody against AF I revealed the presence of the AF II-epitope in the intestine, whereas the AF I-epitope was found in the hypodermis, contractile zone of somatic muscle cells and the external musculature of the uterus. To our knowledge, this is the first report of the presence of a sulfatide in an invertebrate.      

Aromatherapy on a large scale: exposing entire adult holding rooms to ginger root oil increases the mating competitiveness of sterile males of the Mediterranean fruit fly in field cage trials

The sterile insect technique (SIT) is widely used in integrated programs against fruit fly pests, particularly the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in male mating competitiveness. One potential solution involves the pre-release exposure of males to specific attractants. In particular, male exposure to ginger root oil [Zingiber officinale Roscoe (Zingiberaceae); hereafter GRO] has been shown to increase mating success dramatically in field cage trials. Initial studies exposed small groups of males (25 individuals), but more recent work has demonstrated that GRO exposure involving standard storage boxes (containing ≈ 36 000 males) also results in enhanced mating performance. The objective of the present study was to determine whether aromatization of entire trailers, holding ≈ 14 million sterile males from a genetic sexing [temperature sensitive lethal (tsl)] strain, increases male mating success. Independent of the total dose, spatial distribution, or type of dispenser used, sterile males exposed to GRO for a 24-h period displayed greater mating success than non-exposed males in mating cage trials (in which tsl males competed against males from a standard, bisexual strain for females from this same standard strain). Averaged over all experiments, tsl males exposed to GRO obtained 54% of all matings compared to 38% for non-exposed tsl males, an increase of 42%. The implications of these findings for SIT programs against C. capitata are discussed.     

Use of shape to distinguish Chamelea gallina and Chamelea striatula (Bivalvia: Veneridae): Linear and geometric morphometric methods

The commercially fished striped venus clams Chamelea gallina and C. striatula (Bivalvia: Veneridae) are difficult to distinguish by inexperienced observers and the taxonomy of these species is still an issue of discussion. The differences in shape between C. gallina and C. striatula from Portuguese coastal waters were studied through conventional linear and geometric morphometric analysis, using both contour (elliptic Fourier analysis) and landmark-based methods. The relationships shell length vs. height, width, and total weight were significantly different between species. However, because there was a considerable overlap in the data sets, the species could not be distinguished using any combination of those linear measurements. Geometric morphometric methods provided shape variables that led to 0-6% misclassification rates between species; linear morphometric measures led to 16.8% error. Contour analysis revealed differences primarily in the shell umbo and lunular area. The umbo was more "sharp" and the lunula less pronounced in C. striatula than in C. gallina. Generalized procrustes superimposition (landmark analysis) showed that the main differences between species reside in the length of the pallial sinus. Thus, an index was developed (PI: Pallial Index = pallial sinus length/shell length), which was successfully used to separate the species (with 100% correct classification), i.e., specimens with PI lower than 0.119 belonged to C. gallina, whereas greater PI values were attributed to C. striatula. The use of these geometric morphometric methods allowed the detection of differences in shape between these two species and to develop an easy-to-use identification index. We encourage the development of analogous indices that apply the methods of geometric morphometrics to distinguish between other species whose identification is complicated.     

Serologic evidence of human infection by Francisella tularensis in the population of Castilla y León (Spain) prior to 1997

Prior to an outbreak in Castilla y León in December 1997, tularaemia was practically non-existent in Spain. In this paper we studied the prevalence of antibodies against Francisella tularensis in a representative sample of the population (4825 people) from Castilla y León (Spain) in samples collected before this outbreak. Antibodies against F. tularensis were detected in nine (0.19%) of the 4825 sera, with antibody titres ranging from 1/20 to 1/160. Of these nine sera, one was positive in seroagglutination against Brucella. Seroagglutination against other bacteria (Yersinia enterocolitica O:9 and O:3 and Proteus OX19) was negative in all sera. Seroprevalence of antibodies in females was 0.20% and 0.17% in males; no statistically significant differences were found in prevalence in terms of sex, age or province.