The Oct-2 glutamine-rich and proline-rich activation domains can synergize with each other or duplicates of themselves to activate transcription.

The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain. The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain. Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines. The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain. The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain. Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains. An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes. Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

The angiotensinogen gene is located on mouse chromosome 8.

We have recently identified a cis-acting genetic lesion affecting angiotensinogen gene expression in testis and salivary gland. Accordingly, the angiotensinogen gene was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of mouse angiotensinogen sequences by genomic Southern analysis. In AKXD recombinant inbred mice, the angiotensinogen gene is 2.4 +/- 1.8 centiMorgan from Rn7S-8,a 7S RNA gene located on chromosome 8 (Taylor, B.A., personal communication). However, the segregation of salivary and testicular angiotensinogen expression phenotypes into inbred mouse strains was not concordant with the known chromosome 8 proviruses Emv-2, Mtv-21, Xmv-12 or Xmv-26.

The relaxin gene is located on chromosome 19 in the mouse.

Relaxin is a polypeptide hormone that exerts a variety of physiological effects during pregnancy. To investigate the possibility that known genetic mutations affecting aspects of reproductive physiology result in alterations in the structure or production of relaxin, we have determined the chromosomal location of the mouse gene. The finding of a BamHI restriction fragment length polymorphism in AKR mice enabled us to use recombinant inbred strains to make an assignment to chromosome 19. This was confirmed by Southern analysis of DNA from microcell hybrids.

The function of conserved elements in the promoter of the mouse angiotensinogen gene.

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines.

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25-30% of control values. The specificity of this effect was confirmed by showing no decrease in either beta-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.

The angiotensinogen gene of Swiss mice is closely linked to a retrovirus-like element.

Angiotensinogen is cleaved by renin and angiotensin-converting enzyme to liberate the potent vasocontrictor peptide angiotensin II. We have recently identified a cis-acting genetic lesion associated with high levels of angiotensinogen mRNA in the testis and salivary gland of Swiss mice. To determine the molecular basis of this mutation, the Swiss angiotensinogen gene was cloned, and its structure was compared to that from a low-expressing strain (BALB/c). I show that a retrovirus-like element belonging to the intracisternal A-particle gene family has been inserted 9 kb upstream from the cap site of the Swiss angiotensinogen gene. This intracisternal A-particle, named IAP-Agt, segregated concordantly with angiotensinogen expression phenotypes in CXB recombinant inbred mice. However, genomic Southern analysis showed that IAP-Agt was present in some, but not all, inbred laboratory mouse strains displaying high levels of angiotensinogen gene expression. On the basis of this evolutionary evidence, it is unlikely that IAP-Agt is the cause of the angiotensinogen mutation. It is intriguing that Ren-2, the duplicated mouse renin gene, is expressed to high levels in the male salivary gland and also contains a transposed intracisternal A-particle genome.

Generation of homozygosity at the c-Ha-ras-1 locus on chromosome 11p in an adrenal adenoma from an adult with Wiedemann-Beckwith syndrome.

Generation of homozygosity for the human c-Ha-ras-1 locus on the short arm of chromosome #11 (11p) has been demonstrated for an adrenal adenoma from an adult with Wiedemann-Beckwith syndrome (WBS). This is the first demonstration of loss of somatic heterozygosity for a locus on 11p in an adrenal neoplasm and is the first instance where a tumor of any type, from a patient with WBS, shows loss of heterozygosity in this region of the genome. Generation of homozygosity in an adenoma, rather than a carcinoma, demonstrates that this mechanism is an early event in tumorigenesis rather than a late event associated with tumor progression.

Apoptosis, lymphocytotoxicity and the containment of viral infections.

It is generally agreed that cellular immunity plays an important role in limiting certain primary viral infections. Morphological studies indicate that cell death induced by T cells, K cells and NK cells takes the form of apoptosis, not classical necrosis. Killing of a virus-infected cell by either of these means prior to the assembly of infectious virus would clearly contain the infection. Our hypothesis is that the exclusive involvement of apoptosis in lymphocytotoxicity may have additional advantages in preventing virus dissemination. Firstly, a very early event in apoptosis is activation of endogenous, non-lysosomal endonuclease, and this might destroy virus. Secondly, apoptosis results in the formation of membrane-bounded cell fragments, which are phagocytosed intact and digested within the lysosomes of adjacent cells. In contrast, necrosis is characteristically associated with rupture of the cell membrane and release of cellular contents; its induction by non-budding viruses aids in spread of the infection.

Immobilization-induced hypercalcemia and regional osteoporosis.

Immobilization hypercalcemia accompanied by unusual hormonal and bony changes complicated the Guillain-Barré syndrome in a 21-year-old woman. Metaphyseal rarefaction appeared in many sites and was severe in the lower limbs. A bone scan showed increased uptake of 99mTc methylene diphosphonate at these sites and throughout the axial skeleton. The literature on experimental metaphyseal rarefaction suggests that osteoclastic resorption and enhanced regional blood flow are associated with immobilization. Suppression of this osteoclastic component of the increased bone turnover, especially if it is widespread, was the rationale for treatment with calcitonin (CT). The patient also became amenorrheic, with low plasma gonadotropin and estrogen levels. Estrogen therapy coincided with a return of plasma calcium to normal.

Effects of endotoxin on the histology of intact and athymic mice infected with Plasmodium vinckei petteri.

Apparently healthy intact and athymic mice with low to moderate parasitaemias of P. vinckei petteri are very susceptible to the harmful effects of Endotoxin (LPS). The histological changes seen in such mice after injection of a small dose of LPS closely resemble those seen in mice terminally infected with this parasite. Thus the onset of pathology could be hastened by giving a little LPS. Both groups of intact mice showed foci of hepatic necrosis, severe necrosis in the thymus, and light to moderate necrosis in the germinal centres of the splenic white pulp and Peyer's patches. In contrast liver necrosis was seen in very few of the terminally ill athymic mice and in none of the athymic mice given LPS. Our results imply that the lesions produced by LPS in the liver and lymphoid organs of apparently healthy mice with low to moderate parasitaemias would have eventually developed, without the help of extrinsic LPS, as the parasitaemia rose further and the infection ran its normal fatal course. This would be consistent with an intrinsic LPS-like activity in these terminally infected mice. One possible contributor to the liver necrosis seen in this infection is a T-dependent mediator reported to block enzyme induction. Any proposal for the mechanism of this damage must explain its rarity in athymic mice, its induction by LPS in intact but not athymic mice, and host differences in parasite density at which it occurs.

Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice.

Angiotensinogen is the precursor of the potent vasoactive peptide angiotensin II, and is therefore an important determinant of blood pressure and electrolyte homeostasis. In order to map the tissue-specific and inducible enhancer elements governing angiotensinogen gene expression in transgenic mice, we constructed minigenes containing either 0.75 kb or 4 kb or 5' flanking DNA from the BALB/c angiotensinogen gene. Sequences necessary and sufficient to mediate induction by glucocorticoids, oestrogen and bacterial endotoxin were contained on the minigene bearing 0.75 kb of DNA upstream of the capsite. This construct was also able to confer tissue specificity in the majority of organs producing angiotensinogen. In the testis and salivary gland, differences between the donor (BALB/c) and recipient (Swiss) strains were responsible for the apparently aberrant expression of the minigene constructs. The genetic lesion responsible for these expression polymorphisms has been characterized using recombinant inbred mice. An EcoRI restriction fragment length polymorphism which co-segregates with the angiotensinogen expression phenotypes into many inbred mouse strains is also described.

Molecular cloning of the mouse angiotensinogen gene.

We describe here the cloning, restriction mapping, and sequencing of the mouse angiotensinogen gene. The 5' flanking region contains consensus sequences for several hormone-responsive elements and viral-like enhancers within 750 bp of the cap site. The deduced amino acid sequence shows 87% identity with rat angiotensinogen, but there is a discrepancy in the number of cysteine residues in the mature protein among rat (n = 3), human (n = 4), and mouse (n = 4). Because angiotensinogen is homologous to other members of the serine protease inhibitor family, we aligned the putative reactive center of angiotensinogens from various species. This alignment shows that the inhibitor site in human angiotensinogen is different from its rodent counterpart, but the role of this sequence divergence in the pathogenesis of human disease remains to be established.

Acromegaly first diagnosed in pregnancy: the role of bromocriptine therapy.

We report a case of acromegaly diagnosed in the second trimester of pregnancy. Bromocriptine (7.5 mg per day) corrected visual field defects and suppressed prolactin secretion but did not reduce fasting growth hormone levels. We propose that suppression of physiologic lactotroph hyperplasia by bromocriptine may permit noninvasive management of pituitary adenomas during pregnancy.

Virilizing adrenal adenoma in an adult with the Beckwith-Wiedemann syndrome: paradoxical response to dexamethasone.

An adult woman with Beckwith-Wiedemann syndrome, hemihypertrophy and an androgen-secreting adrenal adenoma is described. She presented with a 7-year history of progressive virilization and was found to have high plasma levels of testosterone and dehydroepiandrosterone (DHEA) sulphate and elevated levels of urinary metabolites of testosterone and its precursors. Administration of dexamethasone was associated with progressive rises in plasma 17 alpha OH progesterone, 11 beta-desoxycortisol, DHEA sulphate, androstenedione and testosterone, together with increased urinary excretion of androsterone, 11 beta OH androsterone, etiocholanolone, DHEA, and 16 alpha OH DHEA. Hormone levels fell to normal following removal of the tumour.