Polyamines regulate expression of the neoplastic phenotype in mouse skin

Elevated polyamine levels are characteristic of many types of neoplastic cells and tissues. We demonstrate that in transgenic mice overexpressing ornithine decarboxylase in skin, changes in tissue polyamine levels, particularly putrescine, control the development and maintenance of the neoplastic phenotype. A specific inhibitor of the transgene, alpha-difluoromethylornithine (DFMO), reversibly blocked the appearance of squamous papillomas after carcinogen treatment. Furthermore, treatment of papilloma-bearing mice with DFMO caused rapid tumor regression, also in a reversible manner. Although the rate of apoptosis in papillomas was unaffected by acute DFMO treatment, tumor cell proliferation was rapidly decreased after drug treatment. Conversely, proliferation of normal epidermal keratinocytes was unaffected by DFMO treatment. The regulatory polyamine in this model appears to be putrescine, the immediate product of ornithine decarboxylase. These results demonstrate that elevated polyamine levels are required for both the development and maintenance of the neoplastic phenotype in skin.     

Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression

The mouse renin locus (Ren-1d) exhibits specific patterns of tissue expression. It is expressed in kidney but not submandibular gland (SMG). This locus contains a negative regulatory element (NRE) and a cAMP responsive element (CRE) that share an overlapping sequence. In the kidney, CRE binding proteins (CREB) and NRE binding proteins (NREB) compete for binding to this sequence, with the CREB having a greater affinity. In the SMG, CREB is inactivated by an inhibitory protein, permitting NREB to bind to the sequence, thus inhibiting Ren-1d expression. We hypothesize that the competition between NREB and CREB for this sequence governs tissue-specific expression of mouse renin. We speculate that this may be a general paradigm that determines tissue-specific gene expression.     

A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.     

Multiple gene duplication and expression of mouse bcl-2-related genes, A1

Atrial reentrant tachycardia (ART) was ablated in an anatomically guided approach. Five patients with ART underwent 2 linear incisions without careful pace or activation mapping. One line was from an atrial activation site earlier than P wave onset to the nearest fixed anatomic conduction barrier, i.e., the inferior vena cava or coronary sinus ostium. The other line was made just above or closely crossed the first line vertically. Mean application time was 29 +/- 19 minutes, and the application energy was 14,001 +/- 12,322 joules. Mean follow-up after ablation was 15 +/- 10 months. Three patients underwent electrophysiologic study three months after and sustained ART was not induced. All patients were free of sustained tachycardia events without antiarrhythmic drugs during the postoperative clinical course. Although anatomically guided ablation for ART requires much time and energy, it is easily and effectively done without careful activation or pace mapping, and is indicated if ablation using activation mapping or entrainment technique fails to cure the ART.     

NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae

Distinct classes of sporulation-specific genes are sequentially expressed during the process of spore formation in Saccharomyces cerevisiae. The transition from expression of early meiotic genes to expression of middle sporulation-specific genes occurs at about the time that cells exit from pachytene and form the meiosis I spindle. To identify genes encoding potential regulators of middle sporulation-specific gene expression, we screened for mutants that expressed early meiotic genes but failed to express middle sporulation-specific genes. We identified mutant alleles of RPD3, SIN3, and NDT80 in this screen. Rpd3p, a histone deacetylase, and Sin3p are global modulators of gene expression. Ndt80p promotes entry into the meiotic divisions. We found that entry into the meiotic divisions was not required for activation of middle sporulation genes; these genes were efficiently expressed in a clb1 clb3 clb4 strain, which fails to enter the meiotic divisions due to reduced Clb-dependent activation of Cdc28p kinase. In contrast, middle sporulation genes were not expressed in a dmc1 strain, which fails to enter the meiotic divisions because a defect in meiotic recombination leads to a RAD17-dependent checkpoint arrest. Expression of middle sporulation genes, as well as entry into the meiotic divisions, was restored to a dmc1 strain by mutation of RAD17. Our studies also revealed that NDT80 was a temporally distinct, pre-middle sporulation gene and that its expression was reduced, but not abolished, on mutation of DMC1, RPD3, SIN3, or NDT80 itself. In summary, our data indicate that Ndt80p is required for expression of middle sporulation genes and that the activity of Ndt80p is controlled by the meiotic recombination checkpoint. Thus, middle genes are expressed only on completion of meiotic recombination and subsequent generation of an active form of Ndt80p.     

Divergent expression of alpha1-protease inhibitor genes in mouse and human

The alpha1-protease inhibitor proteins of laboratory mice are homologous in sequence and function to human alpha1-antitrypsin and are encoded by a highly conserved multigene family comprised of five members. In humans, the inhibitor is expressed in liver and in macrophages and decreased expression or inhibitory activity is associated with a deficiency syndrome which can result in emphysema and liver disease in affected individuals. It has been proposed that macrophage expression may be an important component of the function of human alpha1-antitrypsin. Clearly, it is desirable to develop a mouse model of this deficiency syndrome, however, efforts to do this have been largely unsuccessful. In this paper, we report that aside from the issues of potentially redundant gene function, the mouse may not be a suitable animal for such studies, because there is no significant expression of murine alpha1-protease inhibitor in the macrophages of mice. This difference between the species appears to result from an absence of a functional macrophage-specific promoter in mice.     

A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment

The expression of several Arabidopsis thaliana senescence-associated genes (SAGs) in attached and/or detached leaves was compared in response to age, dehydration, darkness, abscisic acid, cytokinin, and ethylene treatments. Most of the SAGs responded to most of the treatments in a similar fashion. Detachment in darkness and ethylene were the strongest inducers of both SAGs and visible yellowing. Detachment in light was also a strong inducer of SAGs, but not of visible yellowing. The other treatments varied more in their effects on individual SAGs. Responses were examined in both older and younger leaves, and generally were much stronger in the older ones. Individual SAGs differed from the norms in different ways, however, suggesting that their gene products play a role in overlapping but not identical circumstances. Some SAGs responded quickly to treatments, which may indicate a direct response. Others responded more slowly, which may indicate an indirect response via treatment-induced senescence. Four new SAGs were isolated as part of this work, one of which shows strong similarity to late embryogenesis-abundant (Lea) genes.     

IL-1-alpha and TNF-alpha differentially regulate CD4 and Mac-1 expression in mouse microglia

The regulatory effects of the proinflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were investigated on CD4 and Mac-1 expression in mouse microglial cultures. The identity of the microglia in cultures was confirmed by multiple indices including morphology, uptake of acetylated low-density lipoprotein and lectin RCA 120 staining. Microglia growing on a monolayer of astrocytes (astrocyte-supported microglia) were both CD4- and Mac-1 positive (out of 94.5 % Mac-1-positive cells, 85.3% were also CD4 positive). When astrocyte-supported microglia were replated directly onto culture dishes (plate-supported microglia), the percentage of CD4- and Mac-1-positive cells decreased to 12-29 and 20-25% respectively. The addition of IL-1alpha or TNF-alpha to plate-supported microglia led to an upregulation of Mac-1 expression in a time- and dose-dependent manner with different EC50s (0.5 ng/ml for IL-1alpha and 2 ng/ml for TNF-alpha) but exhibited similar time-to-peak responses (over 12 h). The addition of IL-1alpha, but not TNF-alpha, also led to an increase in CD4 expression on plate-supported microglia with a similar dose response and time course. IL-1alpha treatment gave rise to an increase in the level of CD4 mRNA as assessed by RT-PCR. The possibility that cell proliferation was responsible for the observed effects on microglia was excluded by an analysis of 3H-thymidine incorporation. Our results suggest that cultured mouse microglia express CD4 molecules which can be upregulated by IL-1alpha while Mac-1 can be upregulated by both IL-1alpha and TNF-alpha.     

Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae

The pathogenesis of multiple sclerosis (MS) is unknown. Searching for possible toxic factors, it was found that 3-day exposure to heat-treated cerebrospinal fluid (CSF) from MS patients caused apoptotic death of astrocytes and oligodendrocytes, but not fibroblasts, myoblasts, Schwann cells, endothelial cells and neurons, in vitro. CSFs from other inflammatory or non-inflammatory neurological diseases showed no toxicity. Exposure of these glial cells to partially purified MS CSF produced DNA fragmentation, apoptotic bodies, chromatin condensation, cell shrinkage, and changes in the levels of known cytokines. A cytotoxic factor, called gliotoxin, was characterized chromatographically as a stable 17-kDa glycoprotein. Since this protein is highly cytotoxic for astrocytes and oligodendrocytes, it may represent an initial pathogenic factor, leading to the neuropathological features of MS, such as blood-brain barrier involvement and demyelination.     

Concentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.