Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.     

Association of heat shock protein 70 with semen quality in boars

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.     

The mechanics of F-actin microenvironments depend on the chemistry of probing surfaces

To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (> 10(4) rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (approximately 100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.     

Erythrocytes possess an intrinsic barrier to nitric oxide consumption

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.     

Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.     

Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway

Transforming growth factor-beta (TGF-beta) is a potent inducer of apoptosis in Hep 3B cells. This work investigated how hepatitis B virus X protein (HBx) affects TGF-beta-induced apoptosis. Trypan blue exclusion and colony formation assays revealed that HBx increased the ID(50) toward TGF-beta. In the presence of HBx, TGF-beta-induced DNA laddering was decreased, indicating that HBx had the ability to block TGF-beta-induced apoptosis. Furthermore, HBx did not alter the expression levels of type I and type II TGF-beta receptors. HBx did not affect TGF-beta-induced activation of promoter activities of the plasminogen activator inhibitor-1 (PAI-1) gene. These results indicate that HBx interferes with only a subset of TGF-beta activity. In the presence of phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin or LY294002, the HBx-mediated inhibitory effect on TGF-beta-induced apoptosis was alleviated. In addition, the tyrosine phosphorylation levels of the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI 3-kinase) and PI 3-kinase activity were elevated in stable clones with HBx expression. Transactivation-deficient mutants of HBx lost their ability to inhibit TGF-beta-induced apoptosis. Phosphorylation of the p85 subunit of PI 3-kinase and Akt, a downstream target of PI 3-kinase, was not observed in stable clones with transactivation-deficient HBx mutant's expression. Thus, the anti-apoptotic effect of HBx against TGF-beta can be mediated through the activation of the PI 3-kinase signaling pathway, and the transactivation function of HBx is required for its anti-apoptosis activity.     

Oligomerizaiton and fibril asssembly of the amyloid-beta protein

In this chapter, we attempt to analyze the evolution of the amyloid-beta (Abeta) molecular structure from its inception as part of the Abeta precursor protein to its release by the secretases and its extrusion from membrane into an aqueous environment. Biophysical studies suggest that the Abeta peptide sustains a series of transitions from a molecule rich in alpha-helix to a molecule in which beta-strands prevail. It is proposed that initially the extended C-termini of two opposing Abeta dimers form an antiparallel beta-sheet and that the subsequent addition of dimers generates a helical Abeta protofilament. Two or more protofilaments create a strand in which the hydrophobic core of the beta-sheets is shielded from the aqueous environment by the N-terminal polar domains of the Abeta dimers. Once the nucleation has occurred, the Abeta filament grows in length by the addition of dimers or tetramers.     

Radiation inactivation analysis of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings

Radiation inactivation analysis was employed to determine the functional masses of enzymatic activity and proton translocation of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings. The activities of H(+)-pyrophosphatase decayed as a simple exponential function with respect to radiation dosage. D(37) values of 6.9+/-0.3 and 7.5+/-0.5 Mrad were obtained for pyrophosphate hydrolysis and its associated proton translocation, yielding molecular masses of 170+/-7 and 156+/-11 kDa, respectively. In the presence of valinomycin and 50 mM KCl, the functional size of H(+)-pyrophosphatase of tonoplast was decreased, while that of submitochondrial particles remained the same, indicating that they are two distinct types of proton pump using PP(i) as their energy source.