Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Magnetic field effects on radical pair intermediates in bacterial photosynthesis

We have investigated the effects of magnetic fields on the formation and decay of excited states in the photochemical reaction centers of Rhodopseudomonas sphaeroides. In chemically reduced reaction centers, a magnetic field decreases the fraction of the transient state P^F that decays by way of the bacteriochlorophyll triplet state P^R. At room temperature, a 2-kG field decreases the quantum yield of P^R by about 40%. In carotenoid-containing reaction centers, the yield of the carotenoid triplet state which forms via P^R is reduced similarly. The effect of the field depends monotonically on field-strength, saturating at about 1 kG. The effect decreases at lower temperatures, when the yield of P^R is higher. Magnetic fields do not significantly affect the formation of the triplet state of bacteriochlorophyll in vitro, the photooxidation of P-870 in reaction centers at moderate redox potential, or the decay kinetics of states P^F and P^R.The effects of magnetic fields support the view that state P^F is a radical pair which is born in a singlet state but undergoes a rapid transformation into a mixture of singlet and triplet states. A simple kinetic model can account for the effects of the field and relate them to the temperature dependence of the yield of P^R.     

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P1) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state (P⁺I^?) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P⁺ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P⁺I^? (or P^F) lives approx. 15 ns. It decays, in part, into a longer lived state (P^R), which appears to be a triplet state. State P^R decays with an exponential time of approx. 55 μs. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P1. Most of P1 then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P1 or P⁺ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I^? suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.     

The type, amount, location, and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides.

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7',8',-tetrahydro-psi,psi-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1, 2, 7', 8'-tetrahydro-psi,psi-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm (Qx) transition of the bacteriochlorophyll complex.     

Delayed fluorescence from Rhodopseudomonas viridis following single flashes

Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studied using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P⁺) of the primary electron-donor (P) and the photoreduced form (X^?) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P⁺ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P⁺X^?, and (c) upon transfer of an electron from X^? to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P⁺ by cytochromes and the oxidation of X^? by Y, the decay kinetics of delayed fluorescence are identical with those of P⁺X^?, as measured from optical absorbance changes.The main decay route for P⁺X^? under these conditions has a rate-constant of approximately 10³ s^?1. In contrast, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P⁺X^? returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s^?1, at 295 °K and pH 7.8. The decay kinetics of P⁺X^? and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal · mol^?1. We conclude that the main decay route involves tunneling of an electron from X^? to P⁺, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P⁺X^? results in increases of both the energy and the entropy of the system, by 16.6 kcal · mol^?1 and 8.8 cal · mol^?1 · deg^?1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.     

Cytochrome photooxidations in Chromatium chromatophores: Each P870 oxidizes two cytochrome C422 hemes

A single, 20-nsec actinic flash oxidizes all of the P870 in Chromatium chromatophores, but only about one half of the cytochrome C422. A second flash, 1–10 msec later, oxidizes most of the remaining cytochrome. The cytochromes which undergo oxidation on the first and second flashes are indistinguishable with respect to their absorption spectra, their kinetics of oxidation and reduction, and their response to N-methylphenazonium methosulfate (PMS) or continuous actinic illumination. The effect of PMS is to increase the total amount of cytochrome C422 which is in the reduced form in the dark, and which is available for photooxidation. The conclusion is that each P870 reaction center is responsible for the oxidation of two C422 hemes.     

Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.