Production of age-related DNA strand breakage in brain cells of senescence-accelerated prone (SAMP1) mouse.

Amounts of DNA strand breaks were estimated by the proportion of cells without tails (PCWT) and the average lengths of tail momentum (ALTM) in comet images of tissue cells of senescence-accelerated prone (SAMP1) mouse and senescence-accelerated resistant (SAMR1) mouse. The PCWT and ALTM of brain cells from SAMR1 were unchanged from 4 to 15 months of age. In the case of SAMP1 brain cells, the PCWT decreased and the ALTM increased in an age-related manner from 8 to 15 months of age. In the cases of liver and kidney, the PCWT and the ALTM of both SAMP1 and SAMR1 cells showed constant values from 4 to 15 months of ages.     

Studies on antiviral glycosides. Synthesis and biological evaluation of various phenyl glycosides.

A variety of analogues and derivatives of phenyl glycosides were synthesized for examination of their biological activities and of the relationship between structure and antiviral activity. For antiviral activity, a 6-deoxy-6-halogeno-D-glucose residue was most suitable for the carbohydrate moiety and p-alkylphenyl groups for the aglycone moiety. Based on these results, p-(sec-butyl)phenyl 6-chloro-6-deoxy-beta-D-glucopyranoside and p-(sec-butyl)phenyl 6-deoxy-6-iodo-beta-D-glucopyranoside were prepared, and the former compound was found to be the most potent antiviral substance, in this series, against influenza and Herpes simplex virus. The anomeric configuration of phenyl glycosides did not contribute to the antiviral activity.     

Expression of MAEG, a novel basement membrane protein, in mouse hair follicle morphogenesis

We screened for genes specifically expressed in the mesenchymes of developing hair follicles using representational differential analysis; one gene identified was MAEG, which encodes a protein consisting of five EGF-like repeats, a linker segment containing a cell-adhesive Arg-Gly-Asp (RGD) motif, and a MAM domain. Immunohistochemistry showed that MAEG protein was localized at the basement membrane of embryonic skin and developing hair follicles, while MAEG expression diminished at the tip of the hair bud. A recombinant MAEG fragment containing the RGD motif was active in mediating adhesion of keratinocytes to the substratum in an RGD-dependent manner. One of the adhesion receptors recognizing the RGD motif was found to be the alpha8beta1 integrin, the expression of which was detected in the placode close to MAEG-positive mesenchymal cells, but later became restricted to the tip of the developing hair bud. Given its localized expression at the basement membrane in developing hair follicles and the RGD-dependent cell-adhesive activity, MAEG may play a role as a mediator regulating epithelial-mesenchymal interaction through binding to RGD-binding integrins including alpha8beta1 during hair follicle development.     

Probing the role of Asp-120(81) of metallo-beta-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.     

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.     

Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats

A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RII alpha, and RII beta) in P(40) fraction (sedimenting at 40,000g), but not in I(40) fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P(40) fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P(40) but not in I(40) fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes.     

Feral cattle (Bos taurus) on Kuchinoshima Island,southwestern Japan: Their stable ranging and unstable grouping

The ranging and grouping of about 75 feral cattle (Bos taurus), inhabiting a small mountainous island with thick vegetation, were studied. Each individual kept a stable home range for at least one year. The cattle preferred flat and open areas (FO areas) as their ranging center, and each had 1 or 2 FO areas in its home range. Those who shared the same FO areas largely overlapped home ranges; thus a clumped pattern was seen in the home range distribution. The individuals within each clump composed a “home range overlapping group” (HROG). The cattle formed 1–8 individual-sized unstable groups mainly with their own HROG's members. FO areas corresponded to the “required components of the home range” reported elsewhere for other ungulate species. Close social interactions were scarcely observed, and no sexual bias in ranging and grouping was confirmed. The stability in ranging and unstability in grouping are discussed in relation to domestication and management of the cattle herd.     

Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[¹⁸F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [⁸⁹Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [¹⁸F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [⁸⁹Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.     

Short peptide tags increase the yield of <Emphasis Type="Italic">C</Emphasis>-terminally labeled protein

C-Terminal protein labeling allows non-radioactive detection of proteins by using fluorescent puromycin derivatives and cell-free translation systems. However, yields of some labeled proteins are low. Here, we report that the yield of labeled protein mainly depends on the C-terminal amino acid sequence. The short peptide tag sequence, RGAA, at the C-terminus increased not only the labeling efficiency (more than 80%) but also the synthesis yield of labeled proteins. To examine the relationship between the C-terminal amino acid sequence and the yield of labeled proteins, we synthesized C-terminally labeled glutathione S-transferase (GST) containing four identical amino acid residues at the C-terminus. The results demonstrated that 4 × Ala, 4 × His, 4 × Gln, and 4 × Cys produced over 200% of the yield of wild-type GST. In addition, the two Ala residues produced almost the same synthesis activity as 4 × Ala and RGAA. Similar results were obtained with various proteins and cell-free translation systems.