The detection of Escherichia coli DNA in the ancient remains of Lindow Man using the polymerase chain reaction

The polymerase chain reaction has been applied to the detection of Escherichia coli DNA in the upper gut contents of Lindow Man, an Iron Age bog body dated to ca 300 BC . With sets of primers from the uidA and lacZ genes, E. coli DNA could be detected reproducibly. Initial attempts at detecting DNA from freshly voided faeces from a healthy volunteer were unsuccessful due to inhibition of the reaction. Development of a method, based on guanidine thiocyanate and silica extraction and purification of the DNA fragments, facilitated the detection of the E. coli DNA in both freshly voided faeces and the upper gut contents of Lindow Man. These findings indicate that it may be possible to study the existence of infectious diseases in ancient civilizations and to learn more about the evolution of microbes.     

Lipase catalysed synthesis of propanediol monoesters in biphasic aqueous medium

The 1,3-regiospecific lipase from Candida deformanscatalysed the esterification of oleic acid and propanediol in biphasic aqueous/lipid medium without organic solvent. The highest conversion of oleic acid into 1,2-propanediol ester was 74% in 24 h with 6.25 mol/l 1,2-propanediol and 0.08 mol/l oleic acid, and produced 100% monoester. The esterification of 1,3-propanediol converted up to 98% of oleic acid into esters in 24 h (with 7.5 mol/l 1,3-propanediol and 0.08 mol/l oleic acid) and formed 35-90% monoester depending on 1,3-propanediol initial concentration (2.5-10 mol/l).     

Incorporation of monoclonal antibodies into cells by osmotic permeabilization. Effect on cellular metabolism

Incorporation of asparagine synthetase-specific monoclonal antibodies into L5178Y D10/R (L-asparaginase-resistant) murine lymphoma cells by osmotic lysis of pinocytic vesicles was used to evaluate the potential of the technique for macromolecular incorporation for metabolic studies. Nonspecific effects of the incorporation procedure included temporary inhibition of protein and DNA synthesis by 80-85% and a transitory loss of membrane integrity. Cells incorporated with an antibody inhibitory to tumor cell asparagine synthetase showed increased dependence upon an exogenous source of asparagine in the culture medium, while cells incorporated with a control antibody were not affected. These studies demonstrated that incorporation of inhibitory monoclonal antibodies into cells can be used to study the short term metabolic role of specific enzymes; however, the metabolic effects induced by the specific macromolecule must be evaluated within the context of the nonspecific effects caused by the osmotic treatment required for incorporation.     

Orientation of dioxygen bound to cobalt(II) bleomycin-DNA fibers

The molecular oxygen adduct of Co(II)-bleomycin is stable for long periods when bound to salmon sperm DNA at large ratios of polymer to drug. According to ESR studies of orientation of the paramagnetic complex associated with DNA fibers, the oxygen-oxygen bond is restricted to a plane perpendicular to the fiber axis. Thus, one can define three g values for the adduct 2.104, 2.016, and 2.000, one parallel to the fiber axis and two orthogonal to it. There is no change in orientation over the range of 77 K to ambient temperature. Furthermore, there is no difference in results at a series of relative humidities ranging from less than 76% where bulk DNA alone assumes an A conformation to 95% where it is primarily B DNA. A structural model is presented for the geometry of the metal binding domain of O2-Co-bleomycin in relationship to the fiber axis of DNA.     

Skeletal and muscular pelvic morphology of hillstream loaches (Cypriniformes: Balitoridae)

The rheophilic hillstream loaches (Balitoridae) of South and Southeast Asia possess a range of pelvic girdle morphologies, which may be attributed to adaptations for locomotion against rapidly flowing water. Specifically, the connectivity of the pelvic plate (basipterygium) to the vertebral column via a sacral rib, and the relative size and shape of the sacral rib, fall within a spectrum of three discrete morphotypes: long, narrow rib that meets the basipterygium; thicker, slightly curved rib meeting the basipterygium; and robust crested rib interlocking with the basipterygium. Species in this third category with more robust sacral rib connections between the basipterygium and vertebral column are capable of walking out of water with a tetrapod-like lateral-sequence, diagonal-couplet gait. This behavior has not been observed in species lacking direct skeletal connection between the vertebrae and the pelvis. The phylogenetic positions of the morphotypes were visualized by matching the morphological features onto a novel hypothesis of relationships for the family Balitoridae. The morphotypes determined through skeletal morphology were correlated with patterns observed in the pelvic muscle morphology of these fishes. Transitions towards increasingly robust pelvic girdle attachment were coincident with a more anterior origin on the basipterygium and more lateral insertion of the muscles on the fin rays, along with a reduction of the superficial abductors and adductors with more posterior insertions. These modifications are expected to provide a mechanical advantage for generating force against the ground. Inclusion of the enigmatic cave-adapted balitorid Cryptotora thamicola into the most data-rich balitorid phylogeny reveals its closest relatives, providing insight into the origin of the skeletal connection between the axial skeleton and basipterygium.     

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.