Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.     

Intracellular distribution of a speech/language disorder associated FOXP2 mutant

Although a mutation (R553H) in the forkhead box (FOX)P2 gene is associated with speech/language disorder, little is known about the function of FOXP2 or its relevance to this disorder. In the present study, we identify the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. A truncated version of FOXP2 lacking the leu-zip, Zn2+ finger, and forkhead domains that was observed in another patient with speech abnormalities demonstrated an aggregated cytoplasmic localization. Furthermore, FOXP2 (R553H) mainly exhibited a cytoplasmic localization despite retaining interactions with nuclear transport proteins (importin alpha and beta). Interestingly, wild type FOXP2 promoted the transport of FOXP2 (R553H) into the nucleus. Mutant and wild type FOXP2 heterodimers in the nucleus or FOXP2 R553H in the cytoplasm may underlie the pathogenesis of the autosomal dominant speech/language disorder.     

Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.     

The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation

Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.     

The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.     

Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division

BACKGROUND AND AIMS: Plants possess three types of DNA methyltransferase, among which methyltransferase type 1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little information is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis was made of its biochemical properties and cellular localization. METHODS: NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays. Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed in tobacco bright yellow 2 cells. KEY RESULTS: In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1. CONCLUSIONS: The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecular interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell division, and may play an important role in progress through mitosis independently of methylation activity.     

Vascular endothelium-derived factors and arterial stiffness in strength- and endurance-trained men

Arterial stiffness is higher in strength-trained humans and lower in endurance-trained humans. However, the mechanisms underlying these different adaptations are unclear. Vascular endothelium-derived factors, such as endothelin-1 (ET-1) and nitric oxide (NO), play an important role in the regulation of vascular tonus. We hypothesized that endogenous ET-1 and NO participate in the adaptation of arterial stiffness in different types of exercise training. The purpose of this study was to investigate plasma ET-1 and NO concentrations and arterial stiffness in strength- and endurance-trained men. Young strength-trained athletes (SA; n = 11), endurance-trained athletes (EA; n = 12), and sedentary control men (C; n = 12) participated in this study. Maximal handgrip strength in SA and maximal oxygen uptake in EA were markedly greater than in C. Aortic pulse-wave velocity, which is an established index of arterial stiffness, was higher in SA and lower in EA than in C. Additionally, we measured systemic arterial compliance (SAC) using carotid artery applanation tonometry and Doppler echocardiography, because arterial stiffness is a primary determinant of the compliance. SAC was lower in SA and higher in EA compared with that in C. Plasma ET-1 concentrations were higher in SA compared with C and EA. We did not find significant differences in plasma NO concentrations (measured as the stable end product of NO, i.e., nitrite/nitrate). The relationships of plasma ET-1 concentrations to aortic pulse-wave velocity and SAC were linear. These results suggest that differences in endogenous ET-1 may partly participate in the mechanism underlying different adaptations of arterial stiffness in strength- and endurance-trained men.     

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.     

A novel in vitro pharmacokinetic/pharmacodynamic model based on two-compartment open model used to simulate serum drug concentration-time profiles

An in vitro pharmacokinetic/pharmacodynamic perfusion model that simulates a two-compartment open model of serum drug concentration-time profiles following intravenous bolus injection and infusion was developed and mathematically described. In the present apparatus model, flow was kept in a one-way mode to avoid liquid traffic, and the washout effect seen in dilution models was overcome by embedding the tested bacteria in low melting point agarose gel. The validity of the equations and the reproducibility of the apparatus model were ascertained by simulating the concentration-time profiles of cefazolin and fosfomycin by substitution of their pharmacokinetic parameters obtained from humans for the equations. An empirical regimen 1X(q24h) of 1 g with cefazolin administered by intravenous infusion effectively killed a Staphylococcus aureus strain. The same regimen with fosfomycin produced a marked kill-curve with a fosfomycin-susceptible enterohaemorrhagic Escherichia coli O157:H7, whereas considerable regrowth was observed with a resistant strain. These results indicated that the present model was able to provide a convenient and reliable method for evaluating the efficacy of antimicrobial agents administered by intravenous infusion.