The Protein Kinase Cdr2, Related to Nim1/Cdr1 Mitotic Inducer, Regulates the Onset of Mitosis in Fission Yeast

Cdc2–Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation. In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2–Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25. These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1. The temperature-sensitive mutation cdc25–22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators. Here we describe that such a screen has identified cdr2⁺, a gene that has an important role in the mitotic control. Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast. Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations. Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1. This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.     

A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.     

Identification of the Functional Domains of the Telomere Protein Rap1 in Schizosaccharomyces pombe

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 ⁺ have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.     

Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes.

We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The Km for the surface concentration of phosphatidic acid was 0.30 mol%. The enzyme was inhibited by sphingosine and chloropromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.     

Thermistor probe for testing an antipyretic suppository in rabbits

The thermistor probe for estimating the effects of an antipyretic suppository after its administration into the rectum of the rabbit was studied. A thermistor probe with three rubber disk stoppers was confirmed to be able to prevent the leakage of drugs from the rectum of a rabbit restrained in a neck stock. By using this newly devised thermistor probe or the usual thermistor probe without a stopper, the febrile response was determined in rabbits injected with bacterial pyrogen. There was no difference in the ability to detect rectal temperature between the two thermistor probes. From these results, it could be concluded that this newly devised thermistor prove was useful in studying the effects of antipyretic suppositories in rabbits.     

Pyrogenic Principle of the Ribonucleic Acid from the Yeast Candida utilis

Experiments were performed to characterize the pyrogenic principle of ribonucleic acid (RNA) from the yeast Candida utilis. It was shown that ribonuclease hydrolysis of the RNA does not lead to inactivation of the pyrogenicity. Pyrogenicity was, however, destroyed by treatment with sodium deoxycholate. On column chromatography with Biogel under sterile and pyrogen-free conditions, the pyrogenic principle of yeast RNA was eluted together with the RNA. After treatment of the RNA with ribonuclease, it was possible to separate the pyrogenic activity from the RNA (hydrolysis products) to a great extent. Column chromatography of Escherichia coli endotoxin showed that the endotoxin was eluted in the same fractions as the pyrogenic activity of yeast RNA. On the basis of the behavior of the pyrogen, it may very well be that the fever reaction is produced not by the nucleic acid but by pyrogenic contaminants of the RNA preparation.     

Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues.

We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species.     

The effect of aging on cutaneous lipid peroxide levels and superoxide dismutase activity in guinea pigs and patients with burns

Cutaneous lipid peroxide levels and superoxide dismutase (SOD) activity in non-aged and aged guinea pigs were measured between 15 min and 7 days after experimental infliction of burns. Skin burns on non-aged and aged patients were also subjected to these assays. In non-aged guinea pig skin burns, lipid peroxide levels increased from 24 hr to the fourth day after the burn infliction, while SOD activity did not increase but showed a slight decrease 12 hr and 24 hr post-burn. On the other hand, while the aged group showed a more increase in skin lipid peroxide levels compared to that seen in non-aged mice, skin SOD activity began to decrease from 30 min post-burn, the maximum decrease being reached on the second day. The activity did not return to normal by the 7th day. In non-aged patients skin burns showed increases in both lipid peroxide levels and SOD activity, while in aged patients, though they showed a marked increase in lipid peroxide levels, SOD activity remained unchanged. The present study indicated that, although in our recent study, skin SOD activity of healthy elderly people was found to be comparable to that in non-aged individuals, the capacity for induction of SOD activity under oxygen stress differed with age in both guinea pig and human burn sufferers. Furthermore, this induction capacity seemed to vary from species to species.     

Modulation of the immune system by Listeria monocytogenes-mediated gene transfer into mammalian cells

In this study, we established a method for Listeria monocytogenes(Lm)-mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm-mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major(L. major )-infection. The injection of Lm modulated the susceptible host into a resistant state against L. major-infection. A more efficient protective effect was obtained with the injection of IL-12-cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm-injection against L. major-infection observed here appeared to be a result of the activation of the local immune system by the Lm-mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.