Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.     

Expression of mRNA for the t-complex polypeptide-1, a subunit of chaperonin CCT, is upregulated in association with increased cold hardiness in Delia antiqua

Summer-diapause and winter-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), were significantly more cold hardy than nondiapause, prediapause, and postdiapause pupae. Moreover, cold acclimation of nondiapause pupae conferred strong cold hardiness comparable with that of diapause pupae. Differential display analysis revealed that the expression of a gene encoding TCP-1 (the t-complex polypeptide-1), a subunit of chaperonin CCT, in D antiqua (DaTCP-1) is upregulated in the pupae that express enhanced cold hardiness. Quantitative real-time polymerase chain reaction analyses showed that the levels of DaTCP-1 messenger RNA in pupal tissues, brain, and midgut in particular, are highly correlated with the cold hardiness of the pupae. These findings suggest that the upregulation of DaTCP-1 expression is related to enhanced cold hardiness in D antiqua. The upregulation of CCT in response to low temperature in an organism other than the yeast is newly reported.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

Polymorphisms of TNFalpha, IL1beta, and IL1RN genes in chronic obstructive pulmonary disease

It is recognized that genetic factors play a role in the susceptibility to COPD. COPD is characterized by airflow limitation. Chronic inflammation causes small airway disease and parenchymal destruction, leading to the airflow limitation. Polymorphisms in pro-inflammatory cytokine genes may confer a risk for the development of COPD. A case-control association study was performed in Japanese population (88 COPD patients and 61 controls) and Egyptian population (106 patients and 72 controls). Genotype and allele frequencies of the TNFalpha -308 G/A and +489 G/A polymorphisms, the IL1beta -511 C/T, -31 T/C, and +3954 C/T polymorphisms, and a VNTR polymorphism in intron 2 of the IL1RN gene were investigated. In addition, pairwise haplotype frequencies were analyzed. When studied independently, none of the polymorphisms were associated with the development of COPD in both populations. However, in the Egyptian population, the distributions of the haplotype (IL1beta -31 T/C : IL1beta +3954 C/T) were significantly different between the COPD patients and the controls (p(corr)=0.0037). Our findings suggest that this haplotype within the IL1beta gene may be involved in the pathogenesis of COPD and that the genetic factors of COPD susceptibility might be different between different populations.     

Fourier transform infrared spectrum of the secondary quinone electron acceptor Q(B) in photosystem II

Fourier transform infrared difference spectra upon single reduction of the secondary quinone electron acceptor Q(B) in photosystem II (PSII), without a contribution from the electron donor-side signals, were obtained for the first time using Mn-depleted PSII core complexes of the thermophilic cyanobacterium Thermosynechococcus elongatus. The Q(B)(-)/Q(B) difference spectrum exhibited a strong C...O stretching band of the semiquinone anion at 1480 cm(-)(1), the frequency higher by 2 cm(-)(1) than that of the corresponding band of Q(A)(-), in agreement with the previous S(2)Q(B)(-)/S(1)Q(B) spectrum of the PSII membranes of spinach [Zhang, H., Fischer, G., and Wydrzynski, T. (1998) Biochemistry 37, 5511-5517]. Also, several peaks originating from the Fermi resonance of coupled His modes with its strongly H-bonded NH vibration were observed in the 2900-2600 cm(-)(1) region, where the peak frequencies were higher by 7-24 cm(-)(1) compared with those of the Q(A)(-)/Q(A) spectrum. These frequency differences suggest that H-bond interactions of the CO groups, especially with a His side chain, are different between Q(B)(-) and Q(A)(-). Furthermore, a prominent positive peak was observed at 1745 cm(-)(1) in the C=O stretching region of COOH or ester groups in the Q(B)(-)/Q(B) spectrum. The peak frequency was unaffected by D(2)O substitution, indicating that this peak does not arise from a COOH group but probably from the 10a-ester C=O group of the pheophytin molecule adjacent to Q(B). The absence of protonation of carboxylic amino acids upon Q(B)(-) formation in contrast to the previous observation in the purple bacterium Rhodobacter sphaeroides suggests that the protonation mechanism of Q(B) in PSII is different from that of bacterial reaction centers.     

Density functional theory calculations on the dielectric constant dependence of the oxidation potential of chlorophyll: implication for the high potential of P680 in photosystem II

The primary donor chlorophyll (Chl) of photosystem II (PSII), P680, has an extremely high oxidation redox potential (E(ox)) of approximately 1.2 V, which is essential for photosynthetic water oxidation. The mechanism for achieving a high potential such as that of P680 has been one of the central questions in photosynthesis research. Here, we have examined the dielectric constant (epsilon) dependence of the E(ox) of monomer Chl using density functional theory calculations with the polarizable continuum model. The calculated E(ox) of a model Chl compound exhibited a sharp increase with a decrease in epsilon in the relatively low epsilon region (epsilon < 5). In contrast, in the higher-epsilon region, E(ox) was rather insensitive to epsilon and converged to a constant value at very high epsilon values. This tendency in the high-epsilon region explains the experimental E(ox) values of isolated Chl a that have been observed in a relatively narrow range of 0.74-0.93 V. The E(ox) of Chl in an ideal hydrophobic protein was estimated to be approximately 1.4 V at an epsilon value of 2. This value indicates that Chl in a hydrophobic environment originally has a high E(ox) that is sufficient for oxidizing water (E(ox) = 0.88 V at pH 6). On the basis of the reported X-ray crystallographic structures, the protein environment of P680 in PSII was estimated to be more hydrophobic than that of the primary donors in bacterial reaction centers. It is therefore suggested that the low-dielectric environment around P680 is one of the major factors in its very high E(ox), and thus, introducing nonpolar amino acids into the binding pocket of P680 was an important process in the evolution of PSII.     

Site-directed mutagenesis of Thermosynechococcus elongatus photosystem II: the O2-evolving enzyme lacking the redox-active tyrosine D

Site-directed mutagenesis in the photosystem II (PSII) oxygen-evolving enzyme was achieved in the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII from this species is the focus of attention because its robustness makes it suitable for enzymological and biophysical studies. PSII, which lacks the redox-active tyrosine Tyr(D), was engineered by substituting a phenylalanine for tyrosine 160 of the D2 protein. An aim of this work was to engineer a mutant for spectroscopy, in particular, for EPR, on the active enzyme. The Tyr(D)(*) EPR signal was monitored in whole cells (i) to control the expression level of the two genes (psbD(1) and psbD(2)) encoding D2 and (ii) to assess the success of the mutagenesis. Both psbD(1) and psbD(2) could be expressed, and recombination occurred between them. The D2-Y160F mutation was introduced into psbD(1) after psbD(2) was deleted and a His-tag was attached to the CP43 protein. The effects of the Y160F mutation were characterized in cells, thylakoids, and isolated PSII. The efficiency of enzyme function under the conditions tested was unaffected. The distribution and lifetime of the redox states (S(n)() states) of the enzyme cycle were modified, with more S(0) in the dark and no rapid decay phase of S(3). Although not previously reported, these effects were expected because Tyr(D)(*) is able to oxidize S(0) and Tyr(D) is able to reduce S(2) and S(3). Slight changes in the difference spectra in the visible and infrared recorded upon the formation and reduction of the chlorophyll cation P(680)(+) and kinetic measurements of P(680)(+) reduction indicated minor structural perturbations, perhaps in the hydrogen-bonding network linking Tyr(D) and P(680), rather than electrostatic changes associated with the loss of a charge from Tyr(D)(*)(H(+)). We show here that this fully active preparation can provide spectra from the Mn(4)CaO(4) complex and associated radical species uncontaminated by Tyr(D)(*).     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.