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Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO.

The maintenance of a stable extracellular concentration of ionized calcium depends on the integrated function of a number of specialized cells (e.g., parathyroid and certain kidney epithelial cells). We recently identified another G protein-coupled receptor (BoPCaRI) from bovine parathyroid that responds to changes in extracellular Ca2+ within the millimolar range and provides a key mechanism for regulating the secretion of parathyroid hormone. Using an homology-based strategy, we now report the isolation of a cDNA encoding an extracellular Ca2+/polyvalent cation-sensing receptor (RaKCaR) from rat kidney. The predicted RaKCaR protein shares 92% identity with BoPCaR1 receptor and features a seven membrane-spanning domain, characteristic of the G protein-coupled receptors, which is preceded by a large hydrophilic extracellular NH2 terminus believed to be involved in cation binding. RaKCaR cRNA-injected Xenopus oocytes responded to extracellular Ca2+, Mg2+, Gd3+, and neomycin with characteristic activation of inositol phospholipid-dependent, intracellular Ca(2+)-induced Cl- currents. In rat kidney, Northern analysis revealed RaKCaR transcripts of 4 and 7 kb, and in situ hybridization showed localization primarily in outer medulla and cortical medullary rays. Our results provide important insights into the molecular structure of an extracellular Ca2+/polyvalent cation-sensing receptor in rat kidney and provide another basis on which to understand the role of extracellular divalent cations in regulating kidney function in mineral metabolism.

The ability of luteinizing hormone-releasing hormone (LHRH) neurons to express the oncogene c-fos was examined during the estrous cycle in rats. The immunocytochemical localization of the c-fos-encoded antigen, Fos, was coupled with the immunocytochemical localization of LHRH. LHRH neurons showed no Fos immunoreactivity during diestrus-1, diestrus-2, estrus, or the morning of proestrus. However, Fos was expressed in LHRH neurons from 1600 to 2200 hours during proestrus. The timing of onset of Fos expression in LHRH neurons during proestrus suggests a strong correlation with increased LH secretion. Pentobarbital, which blocks the preovulatory LH surge, blocked Fos expression in LHRH neurons, but the LHRH neurons expressed Fos on the following afternoon at the time of the expected delayed LH surge. Not all LHRH neurons expressed Fos during the LHRH surge. Approximately half of the LHRH neurons were activated in the preoptic area and anterior hypothalamus; more anteriorly positioned LHRH neurons did not express Fos, resulting in an overall stimulation of 40% of the LHRH neurons. These data provide direct evidence that stimulation of LHRH neurons during proestrus takes place at the LHRH cell bodies, and identify the specific population of LHRH neurons which are activated.

Oleosins are small hydrophobic abundant proteins localized in the oil bodies of plant seeds. An oleosin gene from the monocotyledonous maize (Zea mays L.) was transferred into the dicotyledonous Brassica napus L. using Agrobacterium-mediated transformation. The maize oleosin gene was placed under the control of either its own promoter/terminator or the promoter/terminator of a Brassica seed storage protein (napin) gene. Southern blot analyses of individual transformed plants suggested that the oleosin gene from either construct was incorporated into the Brassica chromosomes without appreciable structural alterations. The amount of construct incorporated was from 1 to >10 copies per haploid genome, depending on the individual transformant. Maize oleosin mRNA and protein were detected only in the transformants containing the napin gene promoter/terminator constructs; these transformants were studied further. Northern blot analyses of RNA isolated from different tissues and seeds of different developmental stages indicated that the maize oleosin mRNA was present only in the maturing seed. Approximately 1% of the total protein in mature seed was represented by maize oleosin. Subcellular fractionation of the mature seed revealed that 90% or more of the maize oleosin, as well as the Brassica oleosin, was localized in the oil bodies. The results show that a monocotyledonous oleosin possesses sufficient targeting information for its proper intracellular transport in a dicotyledon and also suggest that the napin gene promoter/terminator of Brassica, or equivalent seed storage protein regulatory elements of other plant species, may be used to express genes for the genetic engineering of seed oils.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Sp1 and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element of the herpes simplex virus thymidine kinase promoter, are able to direct Sp1-dependent transcription in vitro. The finding that Sp1 is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Sp1 may play a role in modulating transcription of cellular genes.

The 289R E1A protein of adenovirus transactivates a variety of viral and cellular promoters through protein-protein interactions. In earlier studies, mutational analyses of the E1A transactivating domain identified residues that are critical for transactivation and implied that the zinc finger region of the transactivating domain binds a transcription factor. Also, the E1A activation domain was found to bind to the TATA box binding protein (TBP) in vitro. Here, we tested the significance of the E1A-TBP interaction for E1A transactivation by analyzing the effects of conservative substitutions at each of the 49 residues of the E1A activation domain. Seven of the substitutions significantly diminished TBP binding in vitro. All of these were in the zinc finger region and were defective for transactivation in vivo. The perfect correlation between reduced TBP binding and transactivation argues strongly that a direct interaction between the E1A activation domain and TBP is critical to the mechanism of E1A activation. This genetic analysis leads us to further suggest that another factor, which is limiting, is also necessary for E1A-mediated transactivation.