Involvement of DNA mismatch repair in folate deficiency-induced apoptosis small star,filled

Folate is a critical factor for DNA metabolism and its deficiency is associated with a number of human diseases and cancers. Although it has been shown that folate deficiency induces genomic instability and apoptotic cell death, the underlying mechanism is largely unknown. Given the role of mismatch repair in maintaining genomic integrity, mismatch repair was tested for its involvement in folate deficiency-induced genomic instability and cell death. Cells proficient in mismatch repair were highly sensitive to folate deficiency compared with cells defective in either hMutSalpha or hMutLalpha. Since wild-type cells but not mutant cells underwent apoptosis upon extensive folate depletion, the apoptotic response is dependent on a functional mismatch repair system. Our data also indicate that p53 is required for the folate depletion-induced apoptosis. In vitro biochemical studies demonstrated that hMutSalpha specifically recognized DNA damage induced by folate deficiency, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. We conclude that while the mismatch repair-dependent apoptosis is necessary to protect damaged cells from tumorigenesis, it may damage a whole tissue or organ, as seen in patients with megaloblastic anemia, during extensive folate deficiency.     

Rapid effects of estrogen on G protein-coupled receptor activation of potassium channels in the central nervous system (CNS)

Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding and motivated behaviors. Some of the neurons include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and γ-aminobutyric acid (GABA) neurons. We have elucidated several non-genomic pathways through which the steroid alters synaptic responses in these hypothalamic neurons. We have examined the modulation by estrogen of the coupling of various receptor systems to inwardly-rectifying and small-conductance, Ca²⁺-activated K⁺ (SK) channels using intracellular sharp-electrode and whole-cell recording techniques in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples μ-opioid receptors from G protein-gated inwardly-rectifying K⁺ (GIRK) channels in POMC neurons and GABA_B receptors from GIRK channels in dopamine neurons as manifested by a reduction in the potency of μ-opioid and GABA_B receptor agonists to hyperpolarize their respective cells. This effect is blocked by inhibitors of protein kinase A (PKA) and protein kinase C (PKC). In addition, after 24 h following steroid administration in vivo, the GABA_B/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of ₁-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these preoptic GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of GnRH neurons. These findings indicate a richly complex yet coordinated steroid modulation of K⁺ channel activity in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions.     

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

The PWWP domain is a weakly conserved sequence motif found in > 60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223--357) has been structurally characterized at 1.8 A resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Delta218 and Delta369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Delta218 protein, which includes the PWWP domain, binds DNA more strongly than Delta369, which lacks the PWWP domain.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

A rapid,nongenomic action of glucocorticoids in rat B103 neuroblastoma cells

We report here a new example in which glucocorticoids (GCs) acted in a rapid, nongenomic way. In rat B103 neuroblastoma cells, 5-hydroxytryptamine (5-HT) was found to evoke an immediate rise in intracellular free calcium concentration ([Ca(2+)](i)). Pre-incubation of B103 cells for 5 min with corticosterone (B) or bovine serum albumin-conjugated corticosterone (B-BSA) concentration-dependently (10(-4)-10(-8) M) inhibited the peak increments in [Ca(2+)](i). Cortisol and dexamethasone had a similar effect, while deoxycorticosterone and cholesterol were ineffective. This rapid inhibitory effect of corticosterone could be mimicked by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and abolished completely by PKC inhibitors Ro31-8220 or GF-109203X. Neither pertussis toxin (PTX) nor nuclear GC receptor (GR) antagonist RU38486 influenced the rapid action of B. Our results suggest that GCs can modulate the 5-HT-induced Ca(2+) response in B103 cells in a membrane-initiated, nongenomic, and PKC-dependent manner.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.