Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium.

Vascular endothelium is an important transducer and integrator of both humoral and biomechanical stimuli within the cardiovascular system. Utilizing a differential display approach, we have identified two genes, Smad6 and Smad7, encoding members of the MAD-related family of molecules, selectively induced in cultured human vascular endothelial cells by steady laminar shear stress, a physiologic fluid mechanical stimulus. MAD-related proteins are a recently identified family of intracellular proteins that are thought to be essential components in the signaling pathways of the serine/threonine kinase receptors of the transforming growth factor beta superfamily. Smad6 and Smad7 possess unique structural features (compared with previously described MADs), and they can physically interact with each other, and, in the case of Smad6, with other known human MAD species, in endothelial cells. Transient expression of Smad6 or Smad7 in vascular endothelial cells inhibits the activation of a transfected reporter gene in response to both TGF-beta and fluid mechanical stimulation. Both Smad6 and Smad7 exhibit a selective pattern of expression in human vascular endothelium in vivo as detected by immunohistochemistry and in situ hybridization. Thus, Smad6 and Smad7 constitute a novel class of MAD-related proteins, termed vascular MADs, that are induced by fluid mechanical forces and can modulate gene expression in response to both humoral and biomechanical stimulation in vascular endothelium.

Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis.

We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle. In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC6. These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed.

Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth.

Diverse eukaryotic organisms share developmental transcription factors with homologous DNA-binding domains. We showed that the developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF-1, TEC1, Scalloped/TEF-1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of AbaA, TEC1p, is required for this morphological transition. We provide evidence that TEC1p functions in co-operation with STE12p to induce pseudohyphal development.

SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription.

Yeast cyclic AMP (cAMP)-dependent protein kinase (PKA) activity is essential for growth and cell cycle progression. Dependence on PKA function can be partially relieved by overexpression of a gene, SOK2, whose product has significant homology with several fungal transcription factors (StuA from Aspergillus nidulans and Phd1 from Saccharomyces cerevisiae) that are associated with cellular differentiation and development. Deletion of SOK2 is not lethal but exacerbates the growth defect of strains compromised for PKA activity. Alterations in Sok2 protein production also affect the expression of genes involved in several other PKA-regulated processes, including glycogen accumulation (GAC1) and heat shock resistance (SSA3). These results suggest SOK2 plays a general regulatory role in the PKA signal transduction pathway. Expression of the PKA catalytic subunit genes is unaltered by deletion or overexpression of SOK2. Because homozygous sok2/sok2 diploid strains form pseudohyphae at an accelerated rate, the Sok2 protein may inhibit the switch from unicellular to filamentous growth, a process that is dependent on cAMP. Thus, the product of SOK2 may act downstream of PKA to regulate the expression of genes important in growth and development.

Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development.

When starved for nitrogen, MATa/MAT alpha cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition to pseudohyphal growth. A visual genetic screen, called PHD (pseudohyphal determinant), for S. cerevisiae pseudohyphal growth mutants was developed. The PHD screen was used to identify seven S. cerevisiae genes that when overexpressed in MATa/MAT alpha cells growing on nitrogen starvation medium cause precocious and unusually vigorous pseudohyphal growth. PHD1, a gene whose overexpression induced invasive pseudohyphal growth on a nutritionally rich medium, was characterized. PHD1 maps to chromosome XI and is predicted to encode a 366-amino-acid protein. PHD1 has a SWI4- and MBP1-like DNA binding motif that is 73% identical over 100 amino acids to a region of Aspergillus nidulans StuA. StuA regulates two pseudohyphal growth-like cell divisions during conidiophore morphogenesis. Epitope-tagged PHD1 was localized to the nucleus by indirect immunofluorescence. These facts suggest that PHD1 may function as a transcriptional regulatory protein. Overexpression of PHD1 in wild-type haploid strains does not induce pseudohyphal growth. Interestingly, PHD1 overexpression enhances pseudohyphal growth in a haploid strain that has the diploid polar budding pattern because of a mutation in the BUD4 gene. In addition, wild-type diploid strains lacking PHD1 undergo pseudohyphal growth when starved for nitrogen. The possible functions of PHD1 in pseudohyphal growth and the uses of the PHD screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.

SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast.

Mutations in SHR3 block amino acid uptake into yeast by reducing the levels of multiple amino acid permeases within the plasma membrane. SHR3 is a novel integral membrane protein component of the endoplasmic reticulum (ER). shr3 null mutants specifically accumulate amino acid permeases in the ER; other plasma membrane proteins, secretory proteins, and vacuolar proteins are processed and targeted correctly. Our findings suggest that SHR3 interacts with a structural domain shared by amino acid permeases, an interaction required for permease-specific processing and transport from the ER. Even in the presence of excess amino acids, shr3 mutants exhibit starvation responses. shr3 mutants constitutively express elevated levels of GCN4, and mutant shr3/shr3 diploids undergo dimorphic transitions that result in filamentous growth at enhanced frequencies.

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.

Multidegenerate DNA recognition by the OxyR transcriptional regulator.

The Escherichia coli OxyR protein, a regulator of hydrogen peroxide-inducible genes, is a potent stimulator of transcription in its oxidized form but not in its reduced form. OxyR protein purified in its oxidized form was found to bind four of its non-homologous, functional DNA-binding sites with over 10(6)-fold higher affinity than random DNA sequences. A similarly high DNA binding specificity was observed for the reduced (transcriptionally inactive) form of OxyR, consistent with a model in which the OxyR protein is bound to its recognition sequences even in the absence of an oxidative stress. Alignment of five functional OxyR-binding sites revealed a marked lack of perfectly conserved positions, yet an unusually high number of degenerate homologies (positions at which only two of the four possible base pairs are represented). Methylation interference assays on two OxyR-binding sites showed that OxyR contacts its recognition sequences predominantly at positions of degenerate homology. These results suggest that the OxyR protein specifically recognizes seemingly dissimilar sequences through the use of a multidegenerate recognition code. The chemical basis for a plausible degenerate recognition system is discussed.

In vivo oxidative DNA damage: measurement of 8-hydroxy-2'-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection.

HPLC with electrochemical detection is a highly sensitive and selective method for detecting the oxidatively modified DNA residue oh8dG. By this method, the detection of oh8dG from DNA and urine offers a powerful approach for assessing in vivo oxidative damage. Application of this technique to the detection of oh8dG from DNA permits the quantitation of the steady-state levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oh8dG from normal deoxynucleosides. The quantitation of oh8dG in urine complements the measurement of oh8dG in DNA by estimating cumulative oxidative DNA damage in the body. In addition, the urinary assay provides a noninvasive means of measuring this type of damage in laboratory animals and human populations. Thus, an individual animal or human subject may be monitored over time, possibly under various prooxidant conditions, using oh8dG as a sensitive marker for oxidative DNA damage. This analytical approach may allow one to estimate the exposure of an individual to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, and environmental toxins.

Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.

DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.