Brain-reactive antibodies and disease.

Autoimmune diseases currently affect 5-7% of the world's population; in most diseases there are circulating autoantibodies. Brain-reactive antibodies are present in approximately 2-3% of the general population but do not usually contribute to brain pathology. These antibodies penetrate brain tissue only early in development or under pathologic conditions. This restriction on their pathogenicity and the lack of correlation between serum titers and brain pathology have, no doubt, contributed to a delayed appreciation of the contribution of autoantibodies in diseases of the central nervous system. Nonetheless, it is increasingly clear that antibodies can cause damage in the brain and likely initiate or aggravate multiple neurologic conditions; brain-reactive antibodies contribute to symptomatology in autoimmune disease, infectious disease, and malignancy.

Caspr2-reactive antibody cloned from a mother of an ASD child mediates an ASD-like phenotype in mice.

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

Clinical and neuroradiological differences of paediatric acute disseminating encephalomyelitis with and without antibodies to the myelin oligodendrocyte glycoprotein.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) antibodies have been recently described in children with acute disseminating encephalomyelitis (ADEM), but the clinical and neuroradiological characterisation of this subgroup is lacking. OBJECTIVE: To compare the clinical and neuroradiological features of paediatric ADEM with and without MOG antibodies. METHODS: Clinical course, cerebrospinal fluid (CSF)-, MRI studies, outcome and MOG status of 33 paediatric ADEM prospectively studied were reviewed. RESULTS: MOG antibodies (median 1:2560; range 1:160-1:20 480) were detected in 19 children with ADEM. The majority of children showed a decline of serum MOG-IgG titres over time. Children with MOG antibodies did not differ in their age at presentation, sex ratio, the presence of oligoclonal bands, clinical symptoms or initial severity, apart from a higher CSF cell count (p=0.038), compared with children without MOG antibodies. In addition, further relapsing demyelinating episodes associated with MOG antibodies were observed only in children with MOG antibodies. All 19 children with MOG antibodies had a uniform MRI pattern, characterised by large, hazy and bilateral lesions and the absence of atypical MRI features (eg, mainly small lesions, well-defined lesions), which was significantly different compared to that of children without MOG antibodies (p=0.003; and p=0.032, respectively). In addition, children with MOG antibodies had involvement of more anatomical areas (p=0.035) including the myelon characterised by a longitudinally extensive transverse myelitis (p=0.003), more often a complete resolution of lesions (p=0.036) and a better outcome (p=0.038). CONCLUSIONS: Patients with ADEM with MOG antibodies in our cohort had a uniform MRI characterised by large, bilateral and widespread lesions with an increased frequency of longitudinal extensive transverse myelitis and a favourable clinical outcome in contrast to children lacking MOG antibodies.

Persisting myelin oligodendrocyte glycoprotein antibodies in aquaporin-4 antibody negative pediatric neuromyelitis optica.

BACKGROUND: Recently we showed that antibodies to myelin oligodendrocyte glycoprotein (MOG) can be found in aquaporin-4 (AQP4)-immunoglobulin (IgG) seronegative pediatric and adult patients with definite and high-risk neuromyelitis optica (NMO). OBJECTIVE: The purpose of this study was to describe the clinical characteristics and temporal dynamics of MOG-IgG in AQP4-IgG seronegative pediatric patients presenting with definite NMO. METHODS: Children with definite NMO who were referred for further testing of serum antibodies for AQP4 and MOG with a cell-based assay were included in this study. Clinical disease course, cerebrospinal fluid and magnetic resonance imaging (MRI) studies of these patients were reviewed. RESULTS: Between 2008 and 2012 eight children who fulfilled the diagnostic criteria of definite NMO were recruited. Two children with definite NMO tested positive for AQP4-IgG but were negative for MOG-IgG antibodies. Three children had an absence of AQP4-IgG and MOG-IgG antibodies. Three children with definite NMO had high titers of serum MOG-IgG antibodies (≥1: 160), but no AQP4-directed humoral immune response. Longitudinal analysis of serum samples of the latter three children showed persisting high MOG-IgG titers over time. CONCLUSION: Pediatric patients presenting with clinical symptoms and MRI findings highly suggestive of NMO but with high and persisting MOG-IgG antibody titers are most likely to represent a distinct subgroup of acute demyelinating diseases with important clinical and therapeutic implications.

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata.

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.

Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes.

Prostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE(2). However, only NS398 reduced excretion of thromboxane A(2). In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2-derived PG in pathological renal hemodynamic changes in diabetes.

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.

Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.

Functional antagonism between YY1 and the serum response factor.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Antiestrogens: structure-activity relationships and use in breast cancer treatment.

About 70% of breast tumors express estrogen receptor alpha (ERα), which mediates the proliferative effects of estrogens on breast epithelial cells, and are candidates for treatment with antiestrogens, steroidal or non-steroidal molecules designed to compete with estrogens and antagonize ERs. The variable patterns of activity of antiestrogens (AEs) in estrogen target tissues and the lack of systematic cross-resistance between different types of molecules have provided evidence for different mechanisms of action. AEs are typically classified as selective estrogen receptor modulators (SERMs), which display tissue-specific partial agonist activity (e.g. tamoxifen and raloxifene), or as pure AEs (e.g. fulvestrant), which enhance ERα post-translational modification by ubiquitin-like molecules and accelerate its proteasomal degradation. Characterization of second- and third-generation AEs, however, suggests the induction of diverse ERα structural conformations, resulting in variable degrees of receptor downregulation and different patterns of systemic properties in animal models and in the clinic.

Estrogen receptor expression activates the transcriptional and growth-inhibitory response to retinoids without enhanced retinoic acid receptor alpha expression.

Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain.

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis.

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469

The promoter of the H1zero histone gene contains a DNA element bound by retinoic acid receptors.

Retinoic-acid mediated differentiation of F9 cells is accompanied by an increased transcription of the histone H1zero gene. This increase is an early response after addition of retinoic acid, suggesting a direct effect of the hormone on transcription of the gene. We show now that the promoter of histone H1zero contains a DNA element, localized 531 base-pairs upstream of the cap site, that is composed of a direct repeat of the sequence PuGGTCA separated by eight base-pairs. This element confers retinoic acid responsiveness to a heterologous thymidine kinase promoter in F9 and HeLa cells. Furthermore, the element forms retarded complexes not only with bacterially expressed retinoic acid receptors (RARs) and retinoid X receptors (RXRs), but also with endogenous F9 receptors. Our results suggest therefore that retinoic acid receptors can control the expression of a chromatin structural gene the expression of which is associated with a differentiated phenotype.

Glutathione depletion overcomes resistance to arsenic trioxide in arsenic-resistant cell lines.

Arsenic trioxide (As(2)O(3)) is an effective treatment for acute promyelocytic leukemia (APL), but is less effective against other leukemias. Although the response of APL cells to As(2)O(3) has been linked to degradation of the PML/RARalpha fusion oncoprotein, there is evidence that PML/RARalpha expression is not the only mediator of arsenic sensitivity. Indeed, we found that exogenous expression of PML/RARalpha did not sensitize a non-APL leukemic line to As(2)O(3). To evaluate possible other determinants of sensitivity of leukemic cells to As(2)O(3), we derived two arsenic-resistant NB4 subclones. Despite being approximately 10-fold more resistant to arsenic than their parental cell line, PML/RARalpha protein was still degraded by As(2)O(3) in these cells, providing further evidence that loss of expression of the oncoprotein does not confer arsenic sensitivity. Both arsenic-resistant clones contained high glutathione (GSH) levels, however, and we found that GSH depletion coupled with As(2)O(3) treatment dramatically inhibited their growth. Annexin V-staining and TUNEL analysis confirmed a synergistic induction of apoptosis. In addition, these cells failed to accumulate ROS in response to arsenic treatment, in contrast to their arsenic-sensitive parental cells, unless cotreated with buthionine sulfoximine. While other malignant cells did not show a good correlation between arsenic sensitivity and GSH content, GSH depletion nevertheless sensitized all cell lines examined, regardless of their initial response to arsenic alone. These findings suggest that PML/RARalpha expression is not a determinant of arsenic sensitivity, and further support the coupling of GSH depletion and arsenic treatment as a novel treatment for human malignancies that are unresponsive to arsenic alone.

A G577R mutation in the human AR P box results in selective decreases in DNA binding and in partial androgen insensitivity syndrome.

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the C beta of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.

Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells.

The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR species extracted from cells treated with progestin only. The nuclear 9S-[3H]Org2058-PR resulting from cells exposed to Rap, contained, in addition to the heat shock proteins of 90 kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52 but not FKBP51, although the latter was part of unliganded PR heterocomplex associated with hsp90. These results suggest that Rap and KN62 act upon the PR by distinct mechanisms, with only Rap impeding progestin-induced PR transformation. FKBP51 appeared to dissociate from the receptor heterocomplex, but not from hsp90, after hormone binding to PR in vitro and in vivo, whether in the presence or not of Rap and KN62. Immunoprecipitation experiments distinguished two PR- and glucocorticosteroid (GR)-associated molecular chaperone complexes, containing hsp90 and hsp70 and FKBP52 or FKBP51. Another complex identified in T47D cytosol contained hsp90 and the cyclosporin A-binding cyclophilin of 40 kDa, CYP40, but not hsp70, PR, or GR. These observations support the concept that FKBP51 and FKBP52 can act as regulators of Rap and FK506 activity upon PR and GR-mediated transcription, a mechanism that could be also regulated by type II and/or type IV CaMKs.

Effects of meals and time of day on postural blood pressure responses in young and elderly subjects.

Previous data suggest that postural and postprandial hypotension are common in elderly subjects. This study evaluated the effect of age, meals, and time of day on supine and standing blood pressure (BP) and heart rate in healthy young and elderly subjects. A postural BP protocol was performed on 10 young and 16 elderly subjects during an overnight stay. The protocol included first morning and postprandial readings. The systolic and diastolic BP responses to standing were not significantly different between the two groups and were not affected by time of day. Postprandial recumbent BPs declined significantly only in the older subjects. There was no effect of meals on the BP response to standing. We conclude that healthy elderly subjects have a postprandial decline in BP even when supine. However, the BP response to standing is similar in young and elderly subjects and is not impaired by overnight rest or meals. This suggests that the regulation of BP after meals and with standing may be different.

Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells.

Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.

The mouse creatine kinase paired E-box element confers muscle-specific expression to a heterologous promoter.

E-box elements, with the CANNTG sequence motif, occur in numerous promoters and enhancers. We evaluated the tissue-specific expression properties of the paired murine E-box element from the mouse muscle creatine kinase (MCK) enhancer in a minimal heterologous promoter construct. A 46-bp fragment containing the paired E-box element in its wild-type (wt) configuration conferred high levels of muscle-specific expression in transfected embryonic chicken cell cultures. The expression from this paired E-box element was similar to that of the simian virus 40 (SV40) promoter/enhancer, but a 21-bp fragment containing a single E-box was inactive. We conclude that the paired E-box element from the MCK enhancer is sufficient for high levels of muscle-specific expression when placed upstream from a non-muscle TATA element.

Age-related changes in adenylyl cyclase activity in rat aorta membranes.

Blood vessels from aged animals and humans have impaired relaxation and cAMP production to beta-adrenergic stimulation. Direct activators of adenylyl cyclase (AC) such as forskolin are not affected. We hypothesized that analogous findings would occur in membrane preparations. Aortic media membrane preparations from Fischer 344 rats of four age groups (6 weeks to 24 months) were studied. Basal AC activity increased significantly with age. Forskolin-stimulated activity compared to basal tended to be greater in the 6-week and 6-month preparations compared to the 12- and 24-month preparations. AC activity was assessed in the presence of the G protein activators (GTP, GppNHp, NaF). There was no age-related decrease in responsiveness. The receptor agonists isoproterenol (beta-adrenergic), and PGE-1 (prostaglandin), were studied. There was no significant age-related change in responsiveness over basal activity to either of these agonists. There was a slight, but significant increase in the isoproterenol responsiveness over GTP responsiveness in the 6-week-old animals which also approached significance in the 6-month-old animals, but was not seen in the 12- and 24-month-old animals. These data suggest that using a membrane system to assess age-related changes in beta-adrenergic responsiveness in vascular smooth muscle does not retain the robust differences seen in whole vessels.