SNP detection in transforming growth factor-beta1 gene using bacterial magnetic particles

A single nucleotide polymorphism (SNP) within the transforming growth factor-beta1 (TGF-beta1) gene was detected by hybridization-based method using bacterial magnetic particles (BMPs). TGF-beta1 is commonly associated with a single base change resulting in a Leu(10)-->Pro (T(869)-->C) polymorphism and is a genetic marker for susceptibility to osteoporosis. Short (9 bases) and specific probes were designed to detect SNP in TGF-beta1. Detection probes were immobilized on BMPs using cross-linking reagents. TGF-beta1 PCR products (139 bp) were labeled with the fluorescent dye coumarin and hybridized with detection probes on BMPs. Complementary hybridized targets gave over four times higher fluorescent intensities, compared with one base mismatched hybridizations. The SNP genotype was successfully discriminated using this technique.     

A new bipyrrole and some phenolic constituents in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC)

Isolation and structural elucidation of prune constituents were performed and total 10 compounds were determined by NMR and MS analyses. A novel compound was identified to be 2-(5-hydroxymethyl-2',5'-dioxo-2',3',4',5'-tetrahydro-1'H-1,3'-bipyrrole)carbaldehyde, and 7 phenolic compounds were isolated from prunes for the first time. In addition, antioxidant activity of them was evaluated on the basis of the oxygen radical absorbance capacity (ORAC).     

Endurance treadmill training in rats alters CRH activity in the hypothalamic paraventricular nucleus at rest and during acute running according to its period

Running training on the treadmill increases the resting hypothalamic corticotropin-releasing hormone (CRH) content in rats, though is still unknown whether and how it occurs in the parvocellular region of the hypothalamic paraventricular nucleus (PVN) where is a predominant region of pituitary-adrenal activity and where CRH and arginine vasopressin (AVP) are colocalized. We thus aimed at examining whether treadmill training would alter the CRH and AVP mRNA levels in the PVN at rest and during acute running with different lengths of a training regime. Male Wistar rats were subjected to treadmill running (approximately 25 m/min, 60 minutes/day, 5 times/week) for training regimes of 0, 1, 2 or 4 weeks. All training regimes induced an adrenal hypertrophy. Plasma corticosterone levels before acute running increased with lengthening the training period. Four weeks of training produced a significant increase in the resting CRH, but not AVP, mRNA levels in the PVN though relatively shorter training regimes did not. Acute responses of lactate and ACTH release were reduced after 2 and 4 weeks of training, respectively. The responsive PVN CRH mRNA level to acute running decreased with 4 weeks of training but increased with relatively shorter training regimes. These results indicate that running training changes the PVN CRH biosynthetic activity with the regime lasting for 4 weeks, which follows adaptive changes in adrenal functions. Thus, running training-induced changes in hypothalamic CRH activity would originate from the PVN and be induced according to the training period.     

CD4+ NKT cells,but not conventional CD4+ T cells,are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune-privileged site

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8(+) T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8(+) Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Valpha14(+) NKT (iNKT) cells. The iNKT cell subpopulations are either CD4(+) or CD4(-)CD8(-) double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II(-/-) (class II(-/-)) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4(+) T cells are not needed for the development of efferent CD8(+) T cells. Furthermore, Ab depletion of CD4(+) cells in both wild-type mice (remove both conventional and CD4(+) NKT cells) and class II(-/-) mice (remove CD4(+) NKT cells) abrogated the generation of Tr cells. We conclude that CD4(+) NKT cells, but not the class II molecule or conventional CD4(+) T cells, are required for generation of efferent CD8(+) Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8(+) Tr cells may lead to novel immunotherapy for immune inflammatory diseases.     

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins were investigated using -lactalbumin and lysozyme as model proteins. First, reducing conditions were adjusted so that only one of the four disulfide bonds present in each native protein was cleaved. These three-disulfide (3SS) proteins are known to adopt almost native conformations, yet formed precipitates with a basic peptide, lactoferricin, and heparin and heparin fragment, respectively, at concentrations at which native proteins mixed with these compounds remained clear. The 3SS-lysozyme also formed precipitates in the absence of these ligands. Thus, subtle structural changes could lead to aggregation. Electron microscopy revealed fibrillar structures in the aggregates of extensively reduced proteins in the absence of ligands but not in their presence, which shows that the reduction of disulfide bonds suffices for fibril formation and that ligands inhibit fibril formation.     

Statistical evaluation of conditioning for an elite collegiate tennis player using a single-case design

In an individualized athlete's conditioning program, it is desirable to use techniques of single-case research. However, it remains an unsettled question whether statistical analyses are possible in a single-case design. The purpose of the present study was to evaluate the conditioning of a tennis player by statistical analyses over a season using a single-case design. Two male collegiate tennis players (subjects A and B) were observed independently and monitored by self-monitoring sheets during a 6-month tennis season (off-season, preseason, and in-season) using parameters such as performance readiness and performance. Factor analysis was used to extract the fluctuation components of performance readiness. A randomization test was used to examine the difference between means of performance readiness between Deltaoff-pre, Deltapre-in, and Deltaoff-in seasons. The performance readiness increased significantly (p < 0.05) toward a peak date in subject B (p < 0.05). In conclusion, a randomization test was an effective coaching tool to evaluate the conditioning of a tennis player over a training season.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Synthesis,aggregation, neurotoxicity,and secondary structure of various A beta 1-42 mutants of familial Alzheimer's disease at positions 21-23

Cerebral amyloid angiopathy (CAA) due to amyloid beta (A beta) deposition is a key pathological feature of Alzheimer's disease (AD), especially in some form of familial Alzheimer's disease (FAD) including hereditary cerebral hemorrhage with amyloidosis-Dutch type. A beta mainly consists of 40- and 42-mer peptides (Abeta 1-40 and A beta 1-42), which accumulate in senile plaques of AD brains and show neurotoxicity for cultured nerve cells. We synthesized all variant forms of A beta 1-42 associated with reported FAD, such as A21G (Flemish), E22Q (Dutch), E22K (Italian), E22G (Arctic), and D23N (Iowa) along with three potential mutants by one point missense mutation (E22A, E22D, and E22V) in a highly pure form, and examined their ability to aggregate and their neurotoxicity in PC12 cells. The mutants at positions 22 and 23 showed potent aggregative ability and neurotoxicity whereas the potential mutants did not, indicating that A beta 1-42 mutants at positions 22 and 23 play a critical role in FAD of Dutch-, Italian-, Arctic-, and Iowa-types. However, Flemish-type FAD needs alternative explanation except the aggregation and neurotoxicity of the corresponding A beta 1-42 mutant.