An alternative, human SRC promoter and its regulation by hepatic nuclear factor-1alpha.

The SRC gene encodes the proto-oncogene pp60(c-)(src), a tyrosine kinase implicated in numerous signal transduction pathways. In addition, the SRC gene is differentially expressed, developmentally regulated, and frequently overexpressed in human neoplasia. However, the mechanisms regulating its expression have not been completely explored. Here we describe the isolation of a new distal SRC promoter and associated exon, designated 1alpha, which we mapped to a position 1.0 kilobase upstream of the previously described SRC1A housekeeping promoter. Differential use of these promoters and their associated exons coupled with subsequent splicing to a common downstream exon results in c-Src transcripts with different 5' ends but identical coding regions. Promoter analysis following transient transfections into HepG2 cells mapped the minimal 1alpha promoter to a region 145 bp upstream of the major transcription start site. This region contained a consensus binding site for hepatic nuclear factor-1 (HNF-1), a liver-enriched transcription factor implicated in the regulation of a number of genes in liver, kidney, stomach, intestine, and pancreas. Subsequent mobility shift assays confirmed that HNF-1alpha isoform was the predominant factor interacting with this region of the promoter. Mutation of the HNF-1 site resulted in a dramatic reduction in SRC promoter activity. Cotransfection studies demonstrated the promoter could be strongly transactivated by the HNF-1alpha isoform but not by the related HNF-1beta factor. Consistent with these results, we demonstrated that transcripts originating from the SRC1alpha promoter display a tissue restricted pattern of expression with highest levels present in stomach, kidney, and pancreas. These results indicate that SRC transcriptional regulation is much more complex than previously realized and implicates HNF-1 in both the tissue-specific regulation of the SRC gene in normal tissues and the overexpression of c-Src in certain human cancers.

Transcription of the human c-Src promoter is dependent on Sp1, a novel pyrimidine binding factor SPy, and can be inhibited by triplex-forming oligonucleotides.

The tyrosine kinase pp60(c-src) has been implicated in the regulation of numerous normal physiological processes as well the development of several human cancers. However, the mechanisms regulating its expression have not been addressed. In the present study, we report the presence of two Sp1/Sp3 binding sites and three polypurine:polypyrimidine (Pu:Py) tracts in the c-Src promoter that are essential for controlling expression. We demonstrate that Sp1, but not Sp3, is capable of activating the c-Src promoter and that Sp3 is also capable of inhibiting Sp1-mediated transactivation. The presence of multiple Pu:Py tracts conferred S1 sensitivity on plasmids in vitro, suggesting they are capable of adopting non B-DNA conformations. These tracts specifically bind a nuclear factor we named SPy (Src pyrimidine binding factor), which demonstrates both novel double- and single-stranded binding specificities. Mutations eliminating SPy binding compromised Src transcriptional activity, especially in concert with additional mutations affecting Sp1 binding, suggesting the two factors may cooperate in regulating c-Src expression. Finally, we demonstrate that triplex-forming oligonucleotides designed to target both Sp1 and SPy binding sites can down-regulate c-Src expression in vitro, suggesting a potential therapeutic approach to controlling c-Src expression in diseases where aberrant expression or activity has been documented.

What do pregnant women think about the HIV test? A qualitative study.

Before any new antenatal screening test is introduced, the opinions of pregnant women should be considered. This is particularly relevant with HIV testing. This qualitative study reports the views of 29 women attending an antenatal clinic in a large maternity hospital in Scotland where a trial of different ways of offering HIV testing on a universal, voluntary basis occurred. Women were in favour of a test offer, although they did not necessarily wish to accept testing for themselves. Generally they were more worried about having an unhealthy baby. There was a commonly held view that routine testing would cause less anxiety because it would eliminate the stigma of saying yes to testing. A move towards the HIV test being recommended to pregnant women as opposed to merely offered is likely to be acceptable, would probably increase uptake rates and should therefore be assessed.

Uptake and acceptability of antenatal HIV testing: randomised controlled trial of different methods of offering the test.

OBJECTIVE: To determine the uptake and acceptability of different methods of a universal offer of voluntary HIV testing to pregnant women. DESIGN: Randomised controlled trial involving four combinations of written and verbal communication, followed by the direct offer of a test. The control group received no information and no direct offer of a test, although testing was available on request. SETTING: Hospital antenatal clinic covering most of the population of the city of Edinburgh. SUBJECTS: 3024 pregnant women booking at the clinic over a 10 month period. MAIN OUTCOME MEASURES: Uptake of HIV testing and women's knowledge, satisfaction, and anxiety. RESULTS: Uptake rates were 6% for those in the control group and 35% for those directly offered the test. Neither the style of leaflet nor the length of discussion had an effect on uptake. Significant independent predictors of uptake were a direct test offer; the midwife seen; and being unmarried, previously tested, and younger age. Knowledge of the specific benefits of testing increased with the amount of information given, but neither satisfaction nor anxiety was affected by the type of offer. CONCLUSIONS: The universal offer of HIV testing is not intrusive and is acceptable to pregnant women. A policy of offering the HIV test to all women resulted in higher uptake and did not increase anxiety or dissatisfaction. Uptake depends more on the midwife than the method of offering the test. Low uptake rates and inadequate detection of HIV infection point to the need to assess a more routine approach to testing.

Fat absorption from the small intestine of gnotobiotic chicks.

The small intestine of 3-week-old gnotobiotic chicks was divided into five equal segments for analysis. Since it has been found that most fat absorption occurs in the third and fourth segments, they were selected for study. The major fatty acids in the diet and the feces of chicks were palmitic, stearic, oleic, and linoleic acids. Consequently, these fatty acids were selected for quantitative analyses in the absorption studies. Germ-free chicks were contaminated at 7 days of age with bacteria isolated from the third and fourth intestinal segments, except for the Escherichia coli contaminant, which had been previously isolated from a chicken with air sac disease. There were two different degrees of response by chicks to the bacterial contaminants in this study. For this reason, the chicks were placed retrospectively into two categories according to their response to environmental conditions. The first category (A) contained germ-free chicks and chicks monocontaminated with E. coli, Staphylococcus, or Lactobacillus. The second category (B) was composed of conventional chicks and chicks monocontaminated with Streptococcus faecalis or Clostridium welchii, dicontaminated with S. faecalis and C. welchii, and accidentally polycontaminated with coliforms and anaerobic gram-positive bacilli. Chicks in category A demonstrated increased absorption of palmitic and stearic acids and total fat as compared to category B chicks. Conversely, category B chicks showed slightly increased absorption of oleic and linoleic acids. Thin-layer chromatographic analyses demonstrated that cholesterol esters were affected similarly to fatty acids. The site of absorption of fatty acids, cholesterol esters, and total fat was unchanged, regardless of the environment.