The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Identification of newly isolated Babesia parasites from cattle in Korea by using the Bo-RBC-SCID mice

Attempts were made to isolate and identify Korean bovine Babesia parasite. Blood samples were collected from Holstein cows in Korea, and Babesia parasites were propagated in SCID mice with circulating bovine red blood cells for isolation. The isolate was then antigenically and genotypically compared with several Japanese isolates. The Korean parasite was found to be nearly identical to the Oshima strain isolated from Japanese cattle, which was recently designated as Babesia ovata oshimensis n. var. Haemaphysalis longicornis was the most probable tick species that transmitted the parasite.     

Accumulation of a 31-kDa glycoprotein in association with the expression of embryogenic potential by spinach callus in culture

Calli grown from segments of spinach ( Spinacia oleracea L.) root in the presence of gibberellic acid (GA₃) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA₃ failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA₃ included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.     

Using tolerance induced via the anterior chamber of the eye to inhibit Th2-dependent pulmonary pathology

Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-beta2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses.     

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells

DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. DAP12 is expressed not only in NK cells, but also in myeloid cells. Previously, we reported that DAP12 was likely to be involved in monocyte differentiation to macrophage. In this study, we established the mutant DAP12-M1 transfectants (Y76F-M1) that have mutation at their ITAM motifs. We observed that Y76F-M1 cells could not differentiate to macrophages by stimulation via DAP12, whereas wild type DAP12 transfectants (FDAP-M1) could. Furthermore, we demonstrated that the apoptosis signal mediated by LPS was inhibited in Y76F-M1 cells, but was augmented in FDAP-M1 cells. In contrast to the LPS-mediated apoptosis, the combination of LPS and DAP12 stimulation showed good cell viability in FDAP-M1 cells. Collectively our studies demonstrated that DAP12 has a critical role for macrophage differentiation and LPS induced apoptosis in M1 leukemia cells.