Expression of the human beta-amyloid protein of Alzheimer's disease specifically in the brains of transgenic mice

The human beta-amyloid protein is deposited in senile plaques and in the cerebro-vasculature of people with Alzheimer's disease and Down's syndrome. The precise role of beta-amyloid in Alzheimer's disease pathology is presently unknown. To study the properties of beta-amyloid in vivo, we generated transgenic mice that harbor the gene for the carboxyl-terminal 100 amino acids of the human amyloid precursor protein, beginning with the beta-amyloid region, under control of the JC viral early region promoter. The mRNA is expressed exclusively in brain tissue. Further, we demonstrate increased levels of beta-amyloid immunoreactivity on fixed brain tissue. These animals will be useful as a model to study beta-amyloid deposition and its consequences.     

A biochemical and morphological investigation of alkaline phosphatase and Ca+2-ATPase during initial mineralization in chick embryonic tibia

Biochemical analysis of the enzymes alkaline phosphatase and Ca+2-ATPase in the membrane fraction obtained from chick embryonic tibias showed a positive correlation between the elevated activity of these two enzymes and the onset of mineralization. Histochemistry further showed an increased intensity of alkaline phosphatase at the time of the onset of mineralization. It seems that both of these enzymes are involved in the process of mineralization.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction.

Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl- dependent serotonin uptake, with estimated Km and Vmax values of 436 +/- 19 nm and 3.8 +/- 0.6 x 10-18 mol.cell.min-1, respectively. The Manduca high affinity Na+/Cl- dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 micro m) and cocaine (IC50 = 12.89 micro m). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148-152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 micro m). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1-146/MasSERT106-587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 micro m) compared to the human (IC50 = 0.431 micro m) and Manduca serotonin transporters. The chimera MasSERT1-67/hSERT109-630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 micro m) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.     

Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data

Studies of the hyaluronan (HA) tetrasaccharides are important for understanding hydrogen-bonding in the HA polymer, as they are probably the smallest oligomers in which characteristics of the constituent monosaccharides and the polymer are simultaneously exhibited. Here we present extensive molecular dynamics simulations of the two tetrasaccharides of HA in dilute aqueous solution. These simulations have confirmed the existence of intramolecular hydrogen-bonds between the neighboring sugar residues of HA in solution, as proposed by Scott (1989). However, our simulations predict that these intramolecular hydrogen-bonds are not static as previously proposed, but are in constant dynamic exchange on the sub-nanosecond time-scale. This process results in discrete internal motion of the HA tetrasaccharides where they rapidly move between low energy conformations. Specific interactions between water and intramolecular hydrogen-bonds involving the hydroxymethyl group were found to result in differing conformations and dynamics for the two alternative tetrasaccharides of HA. This new observation suggests that this residue may play a key role in the entropy and stability of HA in solution, allowing it to stay soluble up to high concentration. The vicinal coupling constants3 J NHCH of the acetamido groups have been calculated from our aqueous simulations of HA. We found that high values of 3J NHCH approximately 8 Hz, as experimentally measured for HA, are consistent with mixtures of both trans and cis conformations, and thus3 J NHCH cannot be used to imply a purely trans conformation of the acetamido. The rapid exchange of intramolecular hydrogen-bonds indicates that although the structure is at any moment stabilized by these hydrogen-bonds, no one hydrogen-bond exists for an extended period of time. This could explain why NMR often fails to provide evidence for intramolecular hydrogen-bonds in HA and other aqueous carbohydrate structures.      

Analysis of urinary 8-oxo-7,8-dihydro-purine-2'-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2'-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the 'gold standard' techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.