Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells.

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.

Biosynthetic processing of neu differentiation factor. Glycosylation trafficking, and regulated cleavage from the cell surface.

new differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF. In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium.

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

Relationship between the Bracken Basic Concept Scale and the Differential Ability Scales with an at-risk sample of preschoolers.

This study investigated the relationship between the Bracken Basic Concept Scale and the Differential Ability Scales with 35 at-risk preschoolers between the ages 3-6 and 5-11. A Pearson product-moment correlation coefficient of .70 was obtained between the Bracken Basic Concept Scale Total Test scores and the General Conceptual Ability scores of the Differential Ability Scales. This association supports the use of the Bracken scale to predict intelligence if replicated with a larger sample and specific handicaps. In addition, support for the administration of the more brief Bracken School Readiness Composite compared to using the Bracken Total Test score in the prediction of the Differential Ability Scales General Conceptual Ability score was found.

Relaxation training for NIDDM. Predicting who may benefit.

OBJECTIVE: To examine the benefits of relaxation training for patients with NIDDM and to investigate individual differences that could predict a positive response to relaxation training. RESEARCH DESIGN AND METHODS: Thirty-eight subjects with NIDDM were treated with intensive conventional diabetes therapy after an initial metabolic evaluation and psychological and pharmacological testing. Half were assigned to also receive biofeedback-assisted relaxation training. Treatment effects on GHb levels and glucose tolerance were evaluated after 8 wk. RESULTS: Subjects demonstrated significant improvements in GHb level, but not in glucose tolerance, after 8 wk of intensive conventional treatment. These improvements persisted throughout the follow-up period. However, the group provided with relaxation training did not experience greater improvements on either measure than the group given conventional diabetes treatment only. Within the group that received relaxation training, correlations occurred between the improvements in glucose tolerance after treatment and individual differences in trait anxiety and in the effect of alprazolam on glucose tolerance. Differences in the effects of EPI on glucose tolerance and personality measures of neuroticism and perceived locus of control also appeared to be related to improvements in glucose tolerance after training. CONCLUSIONS: Relaxation training did not confer added benefit over and above that provided by conventional diabetes treatment for patients with NIDDM. Additional research is needed to determine whether the administration of relaxation training to selected patients, especially those who are most responsive to stress, would provide benefits for glucose control that are not achieved by conventional treatment.

Correlation of self-administered alcoholism screening test with hemoglobin-associated acetaldehyde.

Thirty subjects underwent analysis of the Self-Administered Alcoholism Screening Test (SAAST) and hemoglobin-associated acetaldehyde levels (HbAA). Eleven alcoholic individuals reporting for treatment, 10 self-defined "social drinkers" and 10 self-defined "teetotallers" who had consumed less than 6 drinks of ethanol per year ever in their lifetime and had not knowingly had ethanol for 6 months were included in the study. The SAAST scores and HbAA levels correlated with one another (r = .55, p = 0.002). For alcoholic individuals, the mean HbAA level was 180 +/- 64 nm/g Hb and the mean SAAST score was 17.3 +/- 6.3. Both analyses could distinguish the alcoholic from the teetotaller group (p = 0.03 for HbAA and p less than 0.001 for the SAAST) and from the group of social drinkers (p = 0.04 for HbAA and p less than 0.001 for the SAAST). The difference in HbAA levels between the social drinkers and teetotallers was not significant (132 +/- 25 vs. 129 +/- 15 nm/g Hb). After modification of the SAAST analysis for teetotallers, SAAST scores were significantly different between social drinkers and teetotallers (3.5 +/- 2.2 vs. 1.1 +/- 1.2, p = 0.009). We conclude that HbAA and SAAST correlate with each other may be clinically useful in distinguishing alcoholic from nonalcoholic individuals. While the SAAST appears to be the more sensitive test, it requires modification in the case of teetotallers.

DNA polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli.

Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA-, dnaBts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA+ strains. Repair synthesis does not require the uvrA gene product.

Effect of bleomycin on deoxyribonucleic acid synthesis in toluene-treated Escherichia coli cells.

The antibiotic bleomycin stimulates deoxyribonucleic acid (DNA) synthesis in toluene-treated Escherichia coli cells. The increase in synthesis is linear with bleomycin concentration. Bleomycin-stimulated DNA synthesis is independent of replication and dependent on DNA polymerase I. Replication is spared as the DNA polymerase I-dependent DNA synthesis increases. Bleomycin does not appear to have any effect on purified E. coli DNA polymerases I or II. Our results suggest that bleomycin causes nicking of the bacterial chromosome with subsequent DNA synthesis catalyzed by DNA polymerase I.