Estrogen receptor variants in normal human mammary tissue

BACKGROUND: Several estrogen receptor (ER) variant messenger RNAs (mRNAs) have been identified previously in human breast cancer biopsy samples and cell lines. The relative levels of certain ER variant mRNAs have been observed to increase with breast tumor progression. In vitro assays of the function of polypeptides encoded by some of these variant mRNAs have led to speculation that ER variants may be involved in the progression from hormone dependence to independence in breast cancer. PURPOSE: We set out to establish if ER variant mRNAs are present in normal human breast tissues and, if so, to compare levels of these variants between normal and neoplastic human breast tissues. METHODS: Four human breast tissue samples from reduction mammoplasties and five samples from tissue adjacent to breast tumors were analyzed. The tissue samples were confirmed to be normal (i.e., not malignant) by histopathologic analysis. RNA was extracted immediately from adjacent frozen sections. Human breast tumor specimens originally resected from 19 patients were acquired from a tumor bank and processed in the same way as the normal tissue samples. The RNAs were then reverse transcribed and subsequently amplified with the use of the polymerase chain reaction (PCR). PCR primer sets were designed to detect several different exon-deleted ER variants and a truncated ER variant (i.e., clone 4). A semiquantitative PCR-based method was used to determine the relative expression of exon 5- and exon 7-deleted variants to wild-type ER mRNAs in the nine normal tissues and in 19 ER-positive breast tumor tissues. The Mann-Whitney rank sum test (two-sided) was used to determine P values. RESULTS: ER variant mRNAs corresponding to the clone 4 ER truncated variant and to variants deleted in either exon 2, exon 3, exons 2-3, exon 5, or exon 7 were detected in all normal samples. The results were confirmed by restriction enzyme analyses and sequencing of the PCR products. The expression of exon 5-deleted ER variant relative to the wild-type ER mRNA was significantly lower (P< .001) in normal tissue than in tumor tissue. A similar trend was noted for expression of the exon 7-deleted ER variant mRNA; however, the difference did not achieve statistical significance (P= .476). CONCLUSION: Several ER variant mRNAs are present in normal human breast tissue, but the level of expression of some of these variants may be lower in normal tissue than in tumor tissue. IMPLICATION: These data suggest that the mechanisms generating ER variant mRNAs exist in normal breast tissue and may be deregulated in breast cancer tissues. Further investigation of the role of variant ER expression in development and progression of human breast cancer appears warranted.     

Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals

Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.     

Design and characterisation of long-R3-insulin-like growth factor-I muteins which show resistance to pepsin digestion

Site-directed mutagenesis was used to construct pepsin-resistant, single-point mutations of the N-terminal extended IGF-I analogue, long-R3-IGF-I. In order to identify the most susceptible sites, the kinetics of long-R3-IGF-I digestion by purified porcine pepsin were determined. Pepsin initially cleaved the Leu10-Phe11 bond in the N-terminal extension peptide to generate FVN-R3-IGF-I, followed in rapid succession by cleavage at Gln15-Phe16, Tyr24-Phe25, Leu10-Val11 and Met59-Tyr60 in the IGF-I moiety. Single-point mutations at these sites were designed on the basis of the preferred cleavage bonds for pepsin, as well as amino acid substitutions less likely to disturb protein structure. These included Leu10Val, Phe16Ala, Phe25Leu, Asp53Glu and Met59Gln. All five muteins retained growth-promoting activity equivalent to or higher than that of IGF-I. In terms of pepsin susceptibility, Leu10Val and Asp53Glu were degraded as rapidly as the parent long-R3-IGF-I, Met59Gln and Phe25Leu were partially stabilised, and Phe16Ala showed a marked improvement in stability over a wide range of pepsin:substrate ratios. Accordingly, the Phe16Ala mutein, long-R3A16-IGF-I, has potential for oral applications to enhance gastric growth and repair.     

Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue

Peripheral nerve injury may lead to a chronic neuropathic pain state that results from an increase in excitability of central neurons. This central sensitization is mediated via an N-methyl-D-aspartic acid (NMDA) receptor and may involve the production of nitric oxide (NO). As NO is suggested to play a role in nociceptive transmission following nerve injury, we examined for altered NO synthase activity at multiple levels of peripheral and spinal neural tissue in a rat model of neuropathic pain. Peripheral neuropathy was induced in rats (N = 12) by ligation of the left L5 and L6 nerve roots. Six other rats had sham surgery. An ipsilateral decrease in paw withdrawal threshold to mechanical stimuli confirmed the presence of a neuropathic pain state. Samples of the lumbar and thoracic spinal cords, L4, L5, and L6 dorsal root ganglia (DRGs), and the sciatic nerves were obtained from the lesioned and contralateral sides at 2 and 4 weeks after neuropathic surgery (N = 6 per group). In the lumbar spinal cord, a bilateral decrease in nitric oxide synthase (NOS) activity was observed 2 and 4 weeks after neuropathic surgery. NOS activity was increased in the ipsilateral L5 and 6 DRGs 2 weeks following neuropathic surgery. An increase in NOS activity in the DRG may be an early mechanism for inducing more central changes. The bilaterally decreased NOS activity in the lumbar spinal cord may be secondary to a negative feedback mechanism resulting from increased NO production in the spinal dorsal root ganglia. Multiple alterations in expression of NOS activity that occur in both peripheral and central processing may play a role in the pain behavior resulting from peripheral nerve injury. (Preliminary results of these studies have been presented in abstract form at the annual meetings of the Society for Neuroscience, 1994, and the American Society of Anesthesiologists, 1994).     

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.     

Noninvasive determination of cardiac output in patients with severe airflow limitation

The noninvasive measurement of cardiac output (Q) by the Indirect Fick CO2-rebreathing technique requires mixed venous P CO2 (P CO2) to be determined by the rebreathing maneuver, and Pa CO2 to be estimated from end-tidal P CO2 (PET CO2). Previous work has suggested that although P CO2 can be determined, Pa CO2 cannot be accurately estimated in patients with significant airflow limitation. Nineteen patients with cystic fibrosis who had severe airflow limitation (%FEV1, 29.3 +/- 7.12 SD) were studied during steady-state exercise at 50% of their measured maximal work capacity. Estimated Pa CO2 was slightly lower than Pa CO2 measured from blood samples obtained from an indwelling arterial catheter (measured: 45.2 +/- 4.92; estimate: 42.7 +/- 5.68 mm Hg). To calculate arterial blood content, the values derived from Pa CO2, pH, hemoglobin (Hb), and O2 saturation were compared with those derived from PET CO2 and O2 saturation, where (1) pH was assumed to be 7.40 and Hb was measured, and (2) pH was assumed to be 7.40 and Hb was assumed to be 15 g/dl (measured mean pH, 7.34; Hb, 14.4 g/dl). No difference in arterial CO2 content was seen between the three methods (measured: 47.53 +/- 5.17; estimate 1: 49.57 +/- 6.58; estimate 2: 49.12 +/- 6.61 ml/100 ml). As pH and Hb can also affect mixed venous CO2 content, the effect on Q was also assessed. Both estimates fit closely with measured Q (r2=0.77 and 0.76), with intercepts not different from zero and slopes not different from 1, and coefficients of variation of 13.5 and 14.6%. When viewed with regard to the confidence intervals for Q as a function of O2 consumption, Q was altered to a minor extent. We conclude that the use of PET CO2 to estimate Pa CO2 can give reasonable values for Q determined noninvasively in patients with severe airflow limitation.