Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe

We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm.     

Mnd1 is required for meiotic interhomolog repair

BACKGROUND: While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle. Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template. RESULTS: Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis. Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template. Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51. Mnd1 does not preferentially associate with hotspots of recombination. CONCLUSIONS: Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template. The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias. Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair. We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.     

Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.     

Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation

Spermatogenesis is dependent on a small population of stem cells. Although stem cells are believed to expand infinitely, there is little functional evidence regarding whether spermatogonial stem cells can increase in their number. Using the spermatogonial transplantation technique, we evaluated the proliferative potential of spermatogonial stem cells in two models of regeneration. After busulfan injection to deplete stem cells, the surviving stem cells were able to expand by at least 15.8-fold within 2 mo. On the other hand, a serial transplantation study indicated that one transplanted stem cell was able to expand by 3.8- and 12-fold within 2 and 4 mo, respectively. These results provide direct functional evidence for the expansion of stem cells and establish the basis for further characterization of the stem cell self-renewal process.     

Biophysical analyses of the transthyretin variants, Tyr114His and Tyr116Ser, associated with familial amyloidotic polyneuropathy

The familial amyloidotic polyneuropathy is strictly associated with point mutations in the coding region of the transthyretin gene. Here, we focused on the mutations in the monomer-monomer and dimer-dimer interaction site of the transthyretin tetramer. The naturally occurring amyloidogenic Tyr114His (Y114H) and Tyr116Ser (Y116S) variants formed more amyloid fibrils than the wild-type transthyretin, nonamyloidogenic Tyr116Val (Y116V) variant, and other amyloidogenic variants in previous studies. The secondary, tertiary, and quaternary structural stabilities of the Y114H and Y116S variants were compared with those of the wild-type transthyretin and nonamyloidogenic Y116V variant. The unfolding data indicated that the amyloidogenic Y114H and Y116S mutations reduced the stability of the secondary, tertiary, and quaternary structure. Our results also indicated that the unfolding of Y114H and Y116S is less cooperative than that of the wild-type transthyretin. Moreover, the tetramer of the amyloidogenic variants dissociated to the monomer even at pH 7.0, indicating the importance of Tyr114 and Tyr116 in strengthening the contacts between monomers and/or dimers of the transthyretin molecule.     

Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis

The testis is one of several immune-privileged organs and is known for its unique ability to support allogeneic or xenogeneic tissue transplants. We investigated the possibility of deriving offspring from mice that underwent transplantation with allogeneic male germ line stem cells in the testis. Although mature adult mice rejected allogeneic germ cells and were infertile, offspring were obtained by intracytoplasmic germ cell injection using partially differentiated donor cells. In contrast, complete spermatogenesis occurred when allogeneic germ cells were transplanted into immature pup testes. Tolerance induction by monoclonal antibody administration allowed the pup transplant recipients to produce allogeneic offspring by natural mating, whereas no spermatozoa were found in the epididymis of untreated recipients. Thus, these results indicate that a histoincompatible recipient can serve as a "surrogate father" to propagate the genetic information of heterologous male donors.