Kinetic measurement of transient dimerization and dissociation reactions of Arabidopsis phototropin 1 LOV2 domain

Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 +/- 0.4) x 10(-11) m2/s to D of the product (8.0 +/- 0.4) x 10(-11) m2/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50-200 microM. According to the Stokes-Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 x 10(-11) m2/s to 9.8 x 10(-11) m2/s with a time constant of 300 micros. The completely opposite D-change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed.     

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization

Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPzeta/RPTPbeta), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein-coupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.     

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.     

Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis

Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.     

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.     

Histological observation of the urogenital papillae in the bi-directional sex-changing gobiid fish, Trimma okinawae

The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male- and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.     

Prevalence and Clinical Features of Hearing Loss Patients with CDH23 Mutations: A Large Cohort Study

Screening for gene mutations in CDH23, which has many exons, has lagged even though it is likely to be an important cause for hearing loss patients. To assess the importance of CDH23 mutations in non-syndromic hearing loss, two-step screening was applied and clinical characteristics of the patients with CDH23 mutations were examined in this study. As a first screening, we performed Sanger sequencing using 304 probands compatible with recessive inheritance to find the pathologic mutations. Twenty-six possible mutations were detected to be pathologic in the first screening. For the second screening, using the probes for these 26 mutations, a large cohort of probands (n = 1396) was screened using Taqman amplification-based mutation analysis followed by Sanger sequencing. The hearing loss in a total of 52 families (10 homozygous, 13 compound heterogygous, and 29 heterozygous) was found to be caused by the CDH23 mutations. The majority of the patients showed congenital, high frequency involved, progressive hearing loss. Interestingly, some particular mutations cause late onset moderate hearing loss. The present study is the first to demonstrate the prevalence of CDH23 mutations among non-syndromic hearing loss patients and indicated that mutations of the CDH23 gene are an important cause of non-syndromic hearing loss.     

Efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a Nedd4-like ubiquitin ligase

To exit infected cells, human immunodeficiency virus type 1 (HIV-1) exploits the vacuolar protein-sorting pathway by engaging Tsg101 and ALIX through PTAP and LYPx(n)L late assembly (L) domains. In contrast, less-complex retroviruses often use PPxY L domains to recruit Nedd4 family ubiquitin ligases. Although HIV-1 Gag lacks PPxY motifs, we now show that the budding of various HIV-1 L-domain mutants is dramatically enhanced by ectopic Nedd4-2s, a native isoform with a truncated C2 domain. The effect of Nedd4-2s on HIV-1 budding required a catalytically active HECT domain and was specific, since other Nedd4 family proteins showed little activity and an unrelated retrovirus was not rescued. The residual C2 domain of Nedd4-2s was critical for the enhancement of HIV-1 budding and for the association of Nedd4-2s with Gag, as reflected by its incorporation into virus-like particles. Interestingly, the incorporation of Nedd4-2s also depended on its active site, indicating that the ability to form a thioester with ubiquitin was required. These data suggest a novel mechanism by which HIV-1 Gag can connect to cellular budding machinery.     

Thermochemical transformation of glucose to 1,6-anhydroglucose in high-temperature steam

An aqueous solution of glucose was reacted at temperatures from 200 to 400 degrees C under atmospheric pressure using a continuous flow reactor. For reaction temperatures above 300 degrees C, the liquid product yield was not sensitive to the temperature change; on the other hand, below 300 degrees C, it decreased rapidly with decreasing temperature. 1,6-Anhydro-beta-D-glucopyranose (AGP) and 1,6-anhydro-beta-D-glucofuranose (AGF) were the major components in the liquid product. The yields of AGP and AGF were 40% and 19%, respectively, at 360 degrees C and a feed rate of 0.5 mL/min. The optimum space time to produce AGP and AGF was about 0.2-0.4s under the present temperature conditions.