Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Design of modified oligodeoxyribonucleotide probes to detect telomere repeat sequences in FISH assays.

A series of dye-labeled oligonucleotide probes containing base and sugar modifications were tested for the ability to detect telomeric repeat sequences in FISH assays. These modified oligonucleotides, all 18 nt in length, were complementary to either the cytidine-rich (C(3)TA(2))(n)or guanosine-rich (T(2)AG(3))(n)telomere target sequences. Oligonucleotides were modified to either increase target affinity by enhancing duplex stability [2'-OMe ribose sugars and 5-(1-propynyl)pyrimidine residues] or inhibit the formation of inter- or intramolecular structures (7-deazaguanosine and 6-thioguanosine residues), which might interfere with binding to the target. Several dye-labeled oligonucleotide probes were found that could effectively stain the telomeric repeat sequences of either cytidine- or guanosine-rich strands in a specific manner. Such probes could be used as an alternative to peptide nucleic acids for investigating the dynamics of telomere length and maintenance. In principle, these relatively inexpensive and readily synthesized modified oligonucleotides could be used for other FISH-related assays.

A phase I/II study of trichosanthin treatment of HIV disease.

Trichosanthin, a ribosomal inhibitor protein, blocks HIV replication in lymphocytes and macrophages. This agent was used to treat 51 patients with advanced HIV disease in a dose-escalation study in which three injections were administered over a 9-21-day period in a dose range of 10-30 micrograms/kg per injection. The maximum tolerated dose was estimated to be 30 micrograms/kg. Reversible but severe fatigue and myalgias were the major dose-limiting side-effects; mild leucocytosis and elevations in serum transaminases were noted and were reversible. Non-dose-related reversible mental status changes were seen in six patients and were considered to be associated with the drug. This was usually manifest as dementia, but progressed to coma in two patients. This reversed, but the sequelae resulted in death in one patient. Decreases in serum p24 antigen levels were noted 1 month after the first infusion in 10 of 18 patients who entered the study with elevated levels; one converted to negative. Values usually remained low to the end of the study period (2 months). In those patients with CD4+ cell levels greater than 50 x 10(6) cells/l significant decreases in sedimentation rate and increases in CD4+ cell numbers were also noted. These changes were found at all dose levels but only in patients receiving three infusions.

Oligonucleotide microarray based detection of repetitive sequence changes.

Prior studies of oligonucleotide microarray-based mutational analysis have demonstrated excellent sensitivity and specificity except in circumstances where a frameshift mutation occurs in the context of a short repeated sequence. To further evaluate this circumstance, a series of nucleic acid samples having heterozygous mutations within repetitive BRCA1 sequence tracts was prepared and evaluated. These mutations included single nucleotide insertions and deletions in homopolymer runs, insertions and deletions of trinucleotide repeats, and duplications. Two-color comparative hybridization experiments were used wherein wild type reference and test targets are co-hybridized to microarrays designed to screen the entire BRCA1 coding sequence for all possible sequence changes. Mutations in simulated heterozygote samples were detected by observing relative losses of test target hybridization signal to select perfect match oligonucleotide probes. While heterozygous mutations could be readily distinguished above background noise in 9/19 cases, it was not possible to detect alterations in a poly dA/dT tract, small triplet repeat expansions, and a 10 bp direct repeat. Unexpectedly, samples containing (GAT)(3) triplet repeat expansions showed significantly higher affinity toward specific perfect match probes relative to their wild type counterparts. Therefore, markedly increased as well as decreased test sample hybridization to perfect match probes should be used to raise a suspicion of repetitive sequence changes.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to C57-black mice leads to parallel decrements in neostriatal dopamine content and tyrosine hydroxylase activity.

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57-black mice (4 doses of 20 mg/kg, at 2 h intervals) led to a large decrement in the neostriatal content of dopamine (92%) and a parallel decrease in tyrosine hydroxylase activity (91%). MPTP administration also caused highly significant but lesser decrements in the neostriatal content of dihydroxyphenylacetic acid (84%) and homovanillic acid (68%). These data are consistent with other observations which suggest that MPTP administration to mice results in a destruction of the dopaminergic nigrostriatal pathway.

Prevention of the nigrostriatal toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by inhibitors of 3,4-dihydroxyphenylethylamine transport.

The 3,4-dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN-35,065-2, WIN-35,428, nomifensine, mazindol, cocaine, McN-5908, McN-5847, and McN-5292 were effective in preventing [3H]DA and [3H]1-methyl-4-phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+-induced release of [3H]DA in vitro. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice (6 X 25 mg/kg i.p.) resulted in a large (70-80%) decrement in neostriatal DA. WIN-35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN-5292 (5 mg/kg), McN-5908 (2 mg/kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP-induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+-induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.

Cocaine blocks the dopamine depletion induced by MPTP.

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice causes a long-lasting depletion of striatal dopamine. The concomitant administration of a dopamine uptake blocker, cocaine, protected the mice from this MPTP-induced dopamine depletion. These observations are discussed in reference to the mechanism through which MPTP enters dopaminergic neurons as well as species differences in the sensitivity of the serotonergic system to the toxic effects of MPTP.

Trichosanthin treatment of HIV-induced immune dysregulation.

Twenty patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC) or asymptomatic HIV infection (HIV+) were given 20 mcg kg-3 trichosanthin (TCS; 'Compound Q'), a ribosome-inactivating protein with in vitro antiviral activity against human immunodeficiency virus (HIV) once every four weeks for up to 12 weeks. With the concurrent administration of prostaglandin inhibitors, the drug was moderately well tolerated, with most subjects experiencing mild arthralgia, hives and malaise. Additionally, four patients experienced neurological complications which resolved spontaneously without intervention. Four of 20 subjects in this open label pilot study showed progressive although transient reductions in viral activity as measured by p-24 antigen level decreases. Subjects also experienced decreases in levels of beta 2-microglobulin. Ten HIV+ and healthy ARC subjects demonstrated improved immunological status as measured by significant increases in percentage of CD4+ cells and augmentations in delayed hypersensitivity reactions. Eight of 20 subjects reported improved appetites and increased energy levels. The group as a whole had a weight gain of 3.2 kg. Eight of 20 subjects who presented with persistent generalized lymphadenopathy exhibited a marked diminution in the size of their lymph nodes after the first treatment. No subject who presented with oral candidiasis experienced an improvement in that condition. We conclude that, in the short term, TCS seems to have the ability to reduce viral activity and improve certain symptoms in healthy ARC patients and HIV + asymptomatics although it may not be able to restore immune competence in persons with advanced AIDS or poor prognosis ARC. Additionally, the drug may pose a special risk for patients with HIV-related dementia.

Normal-incidence grating couplers in Ge-Si.

We report a normal-incidence grating coupler that is monolithically integrated with a Ge(x)Si(1-x) rib waveguide. The design, fabrication, and testing procedures are described for a 0.33-microm-period grating coupler integrated with a Ge(0.04)Si(0.96) waveguide. Also, a theoretical analysis based on coupler-mode theory has been carried out to predict the coupling efficiency and its dependence on the angle of incidence of light. For normal incidence the measured efficiency of 6.5% was found to be in good agreement with the theoretical prediction of 6.9%. The measured FWHM of the acceptance angle about the normal was 5 degrees , compared with the theoretical prediction of 3 degrees .

Thin-film thermo-optic Ge(x)Si(1-x) Mach-Zehnder interferometer.

We report on the design and fabrication of Ge(x)Si(1-x) Mach-Zehnder interferometers based on the thermo-optic effect. The modulators exhibit 8 dB of extinction with a pi-phase-shift power requirement, P(pi), of 245 mW. The 3-dB bandwidth of these thermo-optic devices is 88 kHz. Finally, we present calculations indicating the effect of various parameters on the modulation of the Mach-Zehnder interferometer.