Physical and functional interaction between the Bloom's syndrome gene product and the largest subunit of chromatin assembly factor 1

Bloom's syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.     

Subnucleolar distribution and organization of Vicia faba L. rDNA in situ

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.     

Synthesis, characterization and DNA-binding properties of a new cobalt(II) complex: Co(bbt)2Cl2

A new bis(N-benzyl-benzotriazole)dichloro Co(II) complex (Co(bbt)(2)Cl(2)) was synthesized and the structure was characterized by X-ray crystallography, IR spectrum and elemental analysis. The electrochemical characterization of Co(bbt)(2)Cl(2) was measured in detail, and the interaction of this Co(II) complex with fish sperm DNA was studied by electrochemical techniques and ultraviolet-visible (UV-Vis) spectrophotometry. The cyclic voltammetry (CV) showed that Co(bbt)(2)Cl(2) had two reduction peaks and one oxidation peak on gold electrode. It was found that the currents of both the reduction peaks and the oxidation peak decreased significantly in the presence of DNA compared with those in the absence of DNA, which indicated that Co(bbt)(2)Cl(2) could interact with DNA. The binding of DNA with the complex was not only electrostatic binding but also intercalation.     

Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium

Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Galpha and Gbetagamma subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PH(Crac)-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naive cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PH(Crac)-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PH(Crac)-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity.     

Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model

We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.     

Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.     

A new glycogen synthase activity ratio in skeletal muscle: effects of exercise and insulin

It was recently reported that MnSO4 stimulates glycogen synthase-dependent glucose transfer from UDPglucose into trichloroacetic acid precipitable endogenous glycoproteins (GSMn(T)) in human muscle extracts. To determine the physiologic significance of this reaction, we compared a new GS activity ratio, GSMn(T)/GSH(E) (where GSH(E) represents the usual glucose transfer to ethanol precipitable exogenous glycogen by GS at 7.2 mM glucose 6-phosphate), with the generally used GSL(E)/GSH(E) ratio (where GSL(E) represents glucose transfer at 0.17 mM glucose 6-P concentration). Biopsies were obtained from the quadriceps femoris muscle of healthy subjects at rest, after 40 min of bicycle exercise at approximately 65% of maximal oxygen uptake and after isometric contraction at 2/3 maximal force to fatigue (approximately 1 min). GSMn(T)/GSH(E) increased from 0.012+/-0.002 at rest to 0.054+/-0.008 (P<0.01) after 40 min of bicycle exercise and the increase in GSMn(T) activity was strongly related to the decrease in endogenous glycogen (i.e.. increase in short-chain endogenous glycoproteins) (r=0.90; P<0.05). On the other hand, GSL(E)/GSH(E) did not change significantly after bicycle exercise (rest = 0.49+/-0.04; exercise = 0.58+/-0.08, P>0.05). GSMn(T)/GSH(E) increased from 0.010+/-0.001 at rest to 0.016+/-0.002 (P<0.05) after isometric exercise, whereas GSL(E)/GSH(E) decreased from 0.27+/-0.04 to 0.20+/-0.02 (P<0.05) under corresponding conditions. Last, insulin, which stimulates glycogen synthesis, also increased GSMn(T)/GSH(E) (1.8-fold, P<0.05), as well as GSL(E)/GSH(E) (1.4-fold, P<0.05), in isolated rat soleus muscle. These data indicate that GSMn(T)/GSH(E) is influenced by endogenous substrate availability and covalent modification. Therefore, GSMn(T)/GSH(E) ratio may prove to be a useful alternative to other GS activity ratios that only reflect changes in the phosphorylation state of GS.