Rapid induction of B-cell lymphomas by avian leukosis virus.

Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.

Correlation of antepartum uterine activity and cervical change in twin gestation.

OBJECTIVE: To determine the correlation between quantitative measurements of antepartum uterine activity and cervical change twin gestations. METHODS: Forty women from our Twin Clinic constituted the study group. Participants had a cervical examination each week between 20 and 37 weeks gestation and a cervical score (CS) was calculated as follows: CS = cervical length (cms)-cervical dilation (cms) at the internal os. The women also performed blinded home uterine activity monitoring (HUAM) for a mean of 7.0 + 3.0 hrs/wk (+SD). Uterine activity was expressed as mean number of contractions/hour/week gestation based on the average of three independent reviewers. CS was determined by a single clinician unaware of the HUAM recordings. A significant change in the CS was defined as a reduction of at least 0.5 from the preceding week. Correlation coefficients were used to determine the association between uterine activity and change in the cervical score. RESULTS: Twin pregnancy was characterized by a rise from 0.2 + .03 contractions/hr at 20 weeks to 3.2 + 2.4 contractions/hr at 37 weeks gestation. CS fell from a mean of 2.6 + 0.2 at 20 weeks to -2.1 + 0.9 at 37 weeks gestation. There was a significant negative correlation (-0.317, p < .0001) between increasing uterine activity and decreasing CS. There were significantly more (p < .002) contractions during the 7 days preceding a significant reduction in CS (3.3 + 3.5 contractions/hr) than when the CS was unchanged (1.6 +/- 1.5 contractions/hr). CONCLUSIONS: In twin gestations, an increasing frequency of uterine contractions is strongly correlated with quantifiable cervical change between 20-37 weeks gestation. Persistent daytime contraction frequencies of > 3/hr represent a risk factor for cervical dilation and/or effacement.

Two strong 5' splice sites and competing, suboptimal 3' splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA.

The human immunodeficiency virus type 1 (HIV-1) genome contains 20 exons that are alternatively spliced from 16 splice sites to generate more than 40 different mRNAs, including incompletely spliced and unspliced mRNAs. In contrast to avian retroviral RNA, which has a cis-acting element in gag that negatively regulates splicing (NRS), HIV-1 RNA did not have any NRS sequences in the gag or pol genes detectable by a splicing inhibition assay. However, this assay demonstrated that the HIV-1 first 5' splice site competed with a cellular 5' splice site, suggesting that HIV-1 may have some strong splice sites. To extend this observation, we used a splice site swapping strategy to determine the efficiency of 14 HIV-1 splice sites in human beta globin chimeras tested in transient transfection experiments. While the 1st HIV-1 5' splice site used in all spliced transcripts and the 4th 5' splice site used in most of the 2-kb transcripts were efficient, the other splice sites, including all the 3' splice sites, were less efficient, ranging in use from 25 to 60%. We propose that this range of splice site efficiencies contributes to the regulation of alternative splicing of HIV-1 mRNAs.

Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads.

The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific sequence on the rRNA nascent transcript was labeled in a pattern consistent with its location; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was systematically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simultaneously maintaining sufficient levels of specific labeling.

Proliferative synergy of ANG II and EGF in porcine aortic vascular smooth muscle cells.

To test growth effects of angiotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10(-9) M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (FCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [3H]-thymidine and [35S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% FCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10(3) cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 x 10(-10) M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G1 traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G0/G1 to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").

EGF is incomplete mitogen in porcine aortic smooth muscle cells: DNA synthesis without cell division.

To characterize growth effects of epidermal growth factor (EGF) in subconfluent quiescent porcine aortic vascular smooth muscle cells (VSMC), we measured DNA and protein synthesis by [3H]thymidine (Thd) and [35S]methionine (Met) incorporation, respectively, and cell proliferation rates over 0-6 days in Dulbecco's modified Eagle's-Ham's F-12 media containing 0.4% fetal calf serum (FCS) and insulin. EGF induced dose-dependent [3H]Thd uptake (P less than 0.001); after 10(-9) M EGF, DNA synthesis rate peaked at 24 h, averaging 77% of the response to 10% FCS, and then declined steeply with nadir at 48-60 h. Unexpectedly, EGF failed to induce cell proliferation in the first 4 days, leaving this initial burst of DNA synthesis (12-60 h) uncoupled from cell division. A second lesser but sustained phase of increased DNA synthesis, apparent by day 3-4, was associated with a small increase in cell number on day 6 (P less than 0.05). The early unsustained burst of DNA synthesis reflects EGF's potent mitogenic efficacy for DNA synthesis (G1- to S-phase traversal), probably acting on a subset of cells partially synchronized initially at an EGF-responsive G0/G1 locus; the minimal cell division despite brisk DNA synthesis documents EGF's limited efficacy for (or inhibition of) late cell-cycle events required for completion of mitosis. Late cell-cycle processes are thus rate limiting. EGF also increased protein synthetic rate over control (P less than 0.03) but to a lesser degree (P less than 0.01) than 10% FCS. Indomethacin (10(-6) M) did not alter DNA or proliferative responses to 10(-9) M EGF but transiently augmented EGF-induced protein synthesis (P less than 0.025) at 24 h only.(ABSTRACT TRUNCATED AT 250 WORDS)

Demineralization and remineralization around orthodontic appliances: an in vivo study.

The presence of clinically detectable areas of decalcification (observable as whitened areas) following the removal of orthodontic appliances is well recognized. The aim of the present study was to determine quantitatively the amount of demineralization and the ability of commercially available products to inhibit or reverse orthodontically related demineralization. Twenty orthodontic patients scheduled to have premolars extracted were randomly divided into four groups--one control and three test groups. The extracted premolars (numbering 58) were bracketed using an acid-etch composite system; each patient was given precise oral hygiene instructions and supplied with a sodium fluoride (1,100 ppm fluoride) dentifrice and an orthodontic toothbrush. The control group brushed only with the supplied dentifrice. In addition to brushing with the dentifrice, those in test group I rinsed once each night with a sodium fluoride (0.05%) mouthrinse; group II received a weekly topical APF treatment (1.2% fluoride); and Group III received a weekly topical APF treatment and rinsed once each night with the sodium fluoride mouthrinse. All premolars were extracted after 1 calendar month. Mineral profiles were determined on cross-sectioned teeth 50 to 75 micron occlusal and cervical to the brackets, directly underneath the brackets, and 500 micron away from the brackets. The control teeth (dentifrice only) demonstrated up to 15% demineralization to a depth of 50 micron. All of the test teeth produced rehardening and/or inhibition of demineralization (P less than 0.01). Those in test group III showed a particularly hard outer layer. The study demonstrated that measurable demineralization occurred around orthodontic appliances after only 1 month and this demineralization can be completely inhibited and/or reversed by the use of commercially available fluoride products.