PTEN/MMAC1 mutations and EGFR amplification in glioblastomas.

Loss of heterozygosity (LOH) from chromosome 10 is a hallmark of glioblastoma, the most malignant (grade IV) form of glioma. A candidate tumor suppressor gene, PTEN/MMAC1, that may be targeted for deletion in association with chromosome 10 LOH has recently been identified. Here we have investigated 63 glioblastomas for PTEN/MMAC1 alterations and identified DNA sequence changes that would affect the encoded protein in 17 (27%) tumors. Microsatellite analyses of normal-tumor DNA pairs were performed on 14 of these cases and revealed LOH at locations flanking and/or near PTEN/MMAC1 in all but 1 instance, suggesting that deletion of the remaining wild-type allele had occurred in the large majority of tumors with PTEN/MMAC1 mutations. Competitive PCR assays were developed to address the possible occurrence of PTEN/MMAC1 homozygous deletions in glioblastomas, and this analysis identified three samples having loss of both PTEN/MMAC1 alleles. EGFR amplification was determined to occur at similar frequencies among cases with or without PTEN/MMAC1 homozygous deletions or mutations, suggesting that a growth-promoting effect resulting from amplification-associated increases in epidermal growth factor receptor signaling is not necessarily dependent on the inactivation of PTEN/MMAC1.

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.

A rapid method for quantitating lymphocyte receptor capping: capping defect in AIDS patients.

Capping of concanavalin A (Con A) and anti-Leu-8 (L8) receptors on human peripheral blood mononuclear (PBM) membranes was studied utilizing fluorochrome-conjugated ligands and flow cytometric analysis. Histogram profiles of fluorescent intensities consistently revealed a time-dependent decrease in numbers of brightly fluorescing events concurrent with an increase in numbers of dimly fluorescing events when capping occurs. Differences in fluorescence profiles were detectable by flow cytometric analysis as early as 5 min after capping conditions were initiated. A pronounced defect in receptor capping of PBM cells from AIDS patients was observed. This technique represents a rapid and reproducible means for detecting early changes in cell membrane receptor mobility.

Mecamylamine, but not atropine, antagonizes nicotine-induced hyperglycemia and potentiation of hypnosis produced by pentobarbital in mice.

An anesthetic dose of 50 mg/kg (i.p.) sodium pentobarbital caused a 61% increase in blood glucose levels in mice. Nicotine (2.5 mg/kg) given intraperitoneally 15 min prior to the sodium pentobarbital treatment further increased hyperglycemia by 29% over pentobarbital alone and 90% higher than the control. Mecamylamine (0.5 mg/kg) given intraperitoneally 15 min prior to nicotine resulted in blood glucose concentrations near the control value. Atropine (2 mg/kg) administered intraperitoneally did not prevent the hyperglycemia induced by nicotine and pentobarbital. No significant correlation was observed between the sleep time and the blood glucose of the unconscious or awake mice. However, a significant correlation was observed between the blood glucose concentration of the sleeping and awake mice. Blood glucose levels were always higher when the neuronal activity was depressed and were lower when the neuronal activity was increased.

Nicotine potentiates sodium pentobarbital but not ethanol induced sleep.

Nicotine potentiates, in a dose dependent manner, the sleep time induced by sodium pentobarbital but not by ethanol. Mecamylamine, a nicotinic receptor antagonist, blocked the nicotine induced increase in sleep time. Atropine itself reduced sleep time but did not change the nicotine effect. It is hypothesized that the central and the peripheral nicotinic receptors play an important role in potentiating sodium pentobarbital induced sleep time.

Human immunodeficiency virus gp120 glycoprotein detected by a monoclonal antibody to a synthetic peptide.

A chemically synthesized peptide corresponding to the amino acid sequence 503-532 of gp160 of human immunodeficiency virus (HIV) was used to generate monoclonal antibodies reactive with the env glycoprotein gp120. One monoclonal antibody, 120-1, was isolated that reacted with the peptide and with HIV antigen(s). Western blot analysis showed reactivity with two bands of 120 kDa and 88 kDa. 120-1 reacted in indirect immunofluorescence with 15-20% of infected human T cell line A3.01 as early as 4 days post in vitro infection, 2-3 days prior to detectable reverse transcriptase activity in supernatant fluids.