Transcriptional and post-transcriptional regulation of ß-secretase.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that results in loss of memory and cognitive function, eventually leading to dementia. A key neuropathological event in AD is the cerebral accumulation of senile plaques formed by aggregates of amyloid-ß-peptides (Aß). Aß results from two sequential endoproteolytic cleavages operated on the amyloid-ß precursor protein (AßPP), an integral membrane protein with a single-membrane spanning domain, a large extracellular N-terminus and a shorter, cytoplasmic C-terminus. First, ß-secretase (BACE1) cleaves AßPP at the N-terminal end of the Aß sequence to produce a secreted form of AßPP, named sAßPP, and a C-terminal membrane-bound 99-aminoacid fragment (C99). Then, γ-secretase cleaves C99 within the transmembrane domain to release the Aß peptides of different lengths, predominantly Aß1-40 and Aß1-42.

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure.

The ultraviolet circular dichroism of a protein can be used to estimate the net fraction of its amino acids in different classes of secondary structure. Recent advances in the accuracy of such calculations have resulted from improved computational techniques, as well as extension of the spectral region analyzed to wavelengths less than 180 nm, a wavelength range beyond the limit of most laboratory-based circular dichroism spectrometers. We describe a spectrometer that uses UV radiation from the National Synchrotron Light Source at the Brookhaven National Laboratory to record circular dichroism spectra of proteins (and other biologically important molecules) in aqueous solution over the optimum wavelength range required for calculation of secondary structures. This instrument is available for use by scientists from academic, commercial and research institutions.

Low serum tryptophan to large neutral amino acids ratio in idiopathic infantile autism.

The serum tryptophan to large neutral amino acids ratio (Try/LNAA) is considered a reliable marker of tryptophan availability for brain serotonin synthesis. A dysfunction of brain serotonergic activity has been postulated to exist in autistic disorder and supported by recent studies. On this basis, we determined the serum amino acids levels in 40 children with idiopathic infantile autism as well as in 46 control children. A significantly lower serum Try/LNAA ratio was observed in the autistic subjects compared to the normal controls. In 14 autistic children (35%) this ratio was 2 SD below the mean value obtained in the control group. These results suggest that a low brain tryptophan availability due to a low serum Try/LNAA ratio could be one of the possible mechanisms involved in the alteration of serotonergic function in autism.

Computer network for data acquisition, storage and analysis.

Modern scientific instruments can produce huge quantities of data, usually in digital form. However, data acquisition is only one of three important functions. To be useful, data must also be stored and analyzed. Fortunately, the same computer-based technologies that facilitate the generation of large data-sets provide tools to accomplish these tasks. We describe a data system based on computers connected to a network, developed for this purpose.

Fluorescence of matrix isolated guanine and 7-methylguanine.

We have prepared argon and nitrogen matrices containing guanine and 7-methylguanine, and measured their absorption, fluorescence excitation and fluorescence emission spectra. The fluorescence excitation spectrum of guanine shows four well-resolved bands in the range from 170 to 290 nm; excitation at the wavelengths of each of these bands results in a fluorescence emission with maximum intensity near 350 nm and a single-exponential decay with a lifetime of about 10 ns. There are significant differences between the fluorescent excitation and emission spectra of guanine and of 7-methylguanine, suggesting that the fluorescence observed from the guanine sample does not arise from a minority tautomer.

A mouse repeat sequence conserved in eukaryotic genomes.

To study the biological role of simple repetitive DNA sequences, we analysed a clone isolated from a mouse macrophage cDNA library called T2. This clone contains two simple repetitive sequences: a new sequence, 'CAGAGAGG', and a sequence previously described, 'GATA'. By sequencing analysis, an open reading frame, coding for 225 amino acids, is detected in the T2 clone. The new simple sequence is present thousands of time in the mouse genome, associated or not with the sequence 'GATA'. The sequence 'CAGAGAGG' is transcribed in messenger RNAs. Northern blots of RNAs extracted from adult tissues and from differentiated or non differentiated cell lines show a large number of transcripts that quantitatively decrease when in vitro differentiation occurs. Moreover, Southern blots of DNA extracted from different organisms, hybridize with a fragment containing only the 'CAGAGAGG' sequence, demonstrating that this sequence is represented in the genome of phylogenetically distant eukaryotes, and is highly conserved during eukaryotic evolution.

Identification of a protein encoded by a mouse simple repeated sequence.

In order to study the possibility that a mouse repeated 'simple sequence', containing an ORF, could encode a protein, we inserted a fragment of a cDNA clone into the expression vector pEX31C. The fragment containing the short sequence 'CAGAGAGG' was expressed as MS2 polymerase fusion protein in Escherichia coli. This fusion protein (H5fp) was injected subcutaneously into rabbit and the corresponding polyclonal antibodies generated. Western blot analysis of proteins extracted from different mouse tissues established that anti H5fp antibodies recognized, in vivo, an antigen of 28 KDa. Immunolocalization with anti H5fp antibodies showed the presence of the related antigen in the cytoplasm of the examined sections. An hypothetical secondary structure for the protein was predicted by the Chou and Fasman method.

The integrating ion imager: a device for determining heavy ion doses during irradiations.

We have designed and built an integrating ion imaging system (I3) that records the spatial distribution of the dose of heavy ions incident on samples irradiated at the radiobiology beamline of the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The images of dose are integrated over the duration of the exposure. Unlike the images formed on X-ray film, these images are linear with the incident dose. Heavy ions are incident on a phosphor that is located just behind the sample position. Visible light emitted from the phosphor is collected by a lens and focused onto a scientific grade charge coupled device (CCD) cooled to about -45 degrees C. The phosphor and CCD camera are integral parts of a modular sample holder designed for irradiating molecular samples, which is easily mounted on the sample platform of the beamline. The imager can be adapted to other types of samples. The present CCD image is digitized to 14 bits (16,384 intensity levels), but the dynamic range is extended by adjusting the aperture of the CCD camera lens. Digital images from the CCD are routinely transferred over the BNL local area network for archival storage on a UNIX server, from which they can be opened from any authorized computer with access to the Internet. Images obtained with no sample in place record the dose at all points on the target field. When a sample is in place, an image of the sample appears providing its exact location with respect to fiducial marks recorded for all images. Areas surrounding the image of the sample are used in comparison with companion no-sample images to get exact doses over the sample. The contrast mechanism responsible for image formation is the shift along the Bragg curve resulting from loss of energy of the ions as they pass through the sample--not from a change in ion flux reaching the phosphor. The sharpness of the images formed with the DNA samples we have recorded indicates that neither scattering of the incident heavy ions or the generation of secondary ions contribute significantly.

Clustered DNA damages induced by high and low LET radiation, including heavy ions.

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- ~20%, with the other clustered damages being at least 80%.

Automatic semantic facilitation in Alzheimer's disease.

To explore the nature of semantic deficit in Alzheimer's disease patients (AD patients) we compared two tasks that are known to be very different with respect to the type of attentional demand and conscious effort they require: lexical decision (automatic) in a semantic priming paradigm and semantic relatedness judgements (intentional). In order to minimise post-lexical facilitation we devised a semantic priming experiment that met an automatic condition as much as possible, and we selected patients without severe word recognition deficits. AD patients showed reduced accuracy in the semantic relatedness judgements as compared to controls. Some effect of priming was found, but this was weaker than in normals. AD patients also differed from controls on targets preceded by a nonlinguistic prime (neutral condition) where their reaction times were slower as compared to neutral condition.

Neuropsychological markers of dementia on visual-spatial tasks: a comparison between Alzheimer's type and vascular forms of dementia.

The incidence of the "closing-in" phenomenon and of the tendency to give "primitive answers" on the Raven's Colored Matrices was studied in 50 normal subjects and in two groups of Alzheimer's type (n = 41) and of vascular (n = 35) dementia patients, carefully matched as for the overall severity of dementia and the degree of visual-spatial impairment. The aims of this research were to determine if these patterns of behavior can be considered as neuropsychological markers of dementia and if their incidence is similar in the two dementia groups. Results show that both the closing-in phenomenon and the tendency to give globalistic and odd responses on the Raven's Colored Matrices are good markers of dementia and that, in particular, they point to a degenerative, rather than to a vascular form of dementia. From the clinical point of view, these data suggest that a qualitative analysis of the patient's behavior can increase the diagnostic efficacy of neuropsychological tests and that neuropsychological markers of dementia point more to Alzheimer's disease (considered as the most prototypic form of dementia) than to a vascular form of dementia even when the two groups of patients are well balanced in terms of visual-spatial impairment and the overall severity of dementia.

Two-dimensional, computer-controlled film scanner: quantitation of fluorescence from ethidium bromide-stained DNA gels.

A two-dimensional scanner based on a digital plotter is described. The device is used to analyze photographic negatives of ethidium bromide-stained DNA-agarose gels. Scanning is controlled by and photometric data transferred to a computer for processing, storage, display, and analysis such as integration of the areas under bands and determination of the mean distances of migration of polydisperse samples. An integral light source and detector module designed for reading optical "bar-codes" is mounted in place of the pen of the plotter. Spatial resolution and reproducibility are about 0.2 and 0.005 mm, respectively. Photometric precision as good as one part per thousand is achieved by sinusoidal modulation of the intensity of the light source and synchronous, phase-sensitive detection of the signal from the detector by a lock-in amplifier. No part of the sensor assembly touches the surface of the negative. In contrast to a densitometer, the computer transforms photometric data to values directly proportional to the amount of DNA at given points on the original gel. The ability to move the sensor in two dimensions over the negative allows for the integration across the width of a lane correctly allowing for the nonuniform distribution of the DNA.

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks.

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.

Mechanisms of unilateral spatial neglect in relation to laterality of cerebral lesions.

An Overlapping Figures test, considered as appropriate to study focusing of attention on small but complex stimuli falling in the central parts of visual field and a Searching for Animals test, designed to study the exploration of large parts of extrapersonal space, were administered to 38 controls, and 90 right and 82 left brain-damaged patients. The investigation was designed to test the hypothesis that the extent of space to be explored may have a different influence on unilateral spatial neglect of right and left brain-damaged patients. Both right and left brain-damaged patients showed an asymmetric exploration of space on the Searching for Animals test, making more omissions on the side contralateral to the damaged hemisphere than on the ipsilateral one. On the Overlapping Figures test, however, only right brain-damaged patients showed a clear tendency to omit figures lying on the left side of the composite pattern. This finding suggests that inability to extract visual information from one side of the stimuli during single eye fixations may be the most characteristic feature of unilateral spatial neglect resulting from right hemisphere lesions.

Drugs used in Africa as dyes: I. Skin absorption and tolerability of Bixa orellana L.

Bixa orellana L. (Bixaceae) seed extract, used in Africa as a dye, was employed to evaluate the onset of the possible topical localized responses of the rabbit skin in single application, after repeated application and after UV exposure. Histologic studies were also carried out to evaluate the possible damage and penetration of Bixa colouring matter into the rabbit hairless skin and into the hairs. The study showed that Bixa orellana has a good cutaneous tolerability and may encourage the use as a source of colouring matter in cosmetology.

A comparison of electrophoretic resolution for snapshot and finish-line imaging.

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland, Anal. Biochem. 210, 378-388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish-line imaging provided higher resolving power. The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%.

Neuropsychological abnormalities in AIDS and asymptomatic HIV seropositive patients.

Neuropsychological and immunological parameters were studied in 36 AIDS patients with early disease and without clinical, laboratory, and neuroradiological signs of CNS impairment, and also in 33 asymptomatic HIV seropositive subjects. Many AIDS patients performed abnormally on timed psychomotor tasks, tasks involving sequencing and "set-shifting", and memory tasks stressing attention, learning, active retrieval, and monitoring of information. Asymptomatic HIV seropositive subjects as a group did not perform significantly worse than controls. However, on the basis of a cut off number of pathological performances on neuropsychological tasks, 52.8% of AIDS and 30.3% of asymptomatic HIV seropositive subjects had cognitive impairment, compared with 3.9% of HIV seronegative controls. Low values of CD4+ cells and of CD4+/CD8+ ratio and high titres of P-24 antigen in the blood prevailed among subjects with cognitive impairment, especially in the asymptomatic HIV seropositive group.

Quantifying DNA damage by gel electrophoresis, electronic imaging and number-average length analysis.

DNA damages that can be converted to single- or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge-coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled DNA.