Tektin5, a new Tektin family member, is a component of the middle piece of flagella in rat spermatozoa

Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.     

Fission yeast Swi5 protein, a novel DNA recombination mediator

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: role of NADPH oxidase and mitochondria

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca(2+) influx, while in other cell types ROS are produced in a Ca(2+)-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca(2+) entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca(2+) and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (~2 min). Antigen but not TG caused extracellular release of superoxide (O(2)(*-))/hydrogen peroxide (H(2)O(2)), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca(2+) entry resulted in the generation of O(2)(*-) in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C(4), tumor necrosis factor-alpha, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca(2+) influx by NADPH oxidase and downstream of Ca(2+) influx by the mitochondria regulate the proinflammatory response of mast cells.     

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products

Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcepsilonRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (> or =0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcepsilonRI stimulation increased the cytosolic Ca(2+) levels to a similar extent, whereas GA, but not FcepsilonRI stimulation, caused mitochondrial Ca(2+) overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcepsilonRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca(2+) homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca(2+) overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.     

Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum

Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca(2+) handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species, acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca(2+) handling by measuring Ca(2+) transient using a confocal imaging system and single ryanodine receptor type 2 (RyR2) channel activity using the planar lipid bilayer method. Ca(2+) transient was recorded in isolated rat ventricular myocytes with incorporated fluo 3. Intracellular reduced glutathione level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 microM increased Ca(2+) transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 microM decreased Ca(2+) transient area significantly. Acetaldehyde showed a minor effect on Ca(2+) transient in myocytes in which intracellular reduced glutathione content had been decreased against challenge of diethylmaleate to a level comparable to that induced by exposure to approximately 50 microM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (-213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1-100 microM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (-230 mV) or markedly oxidized (-180 mV) state. This result was similar to effects of acetaldehyde on Ca(2+) transient in diethylmaleate-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.     

Promotion of radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by nitro 5-deazaflavin derivatives.

6-Nitro- and 8-nitro-5-deazaflavin derivatives have been found to enhance prominently the radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at the expense of formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine nucleosides (FapydGuo) both in deaerated and in N(2)O saturated aqueous 2'-deoxyguanosine solutions. The radiosensitizing capacity of a 9-nitro-5-deazflavin derivative was observed only in the N(2)O saturated aqueous solutions.