Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.     

Effect of the energy density of non-purified diets on growth, gestation and lactation in mice

The effects of dietary energy density on the performance of growing, gestating and lactating C57BL/6J mice were determined in order to develop pelleted non-purified practical diets for use in all stages of the mouse life cycle. Experimental diets with 4 levels of energy at 24% crude protein (CP) were pelleted and the nutritional values were determined using adult rats. The nitrogen-corrected metabolizable energy (MEn) values ranged from 2.86 to 3.73 kcal/g and the digestive CP (DCP) contents ranged from 20.5 to 22.6% on a dry matter (DM) basis. Mice responded to decreased dietary energy by increasing their feed intake to maintain MEn intake levels, except for 1 week after weaning and during lactation periods. During these periods, mice fed lower energy diets could not consume as much MEn as those fed higher energy diets. The lowest energy diet, in comparison with the highest energy diet, resulted in approximately a 33% lower weaning weight of pups at 3 weeks of age, a 13.2 to 34.4% slower growth at 3 to 4 weeks of age, and a 9.3 day delay in the onset of vaginal opening in young females. Lower energy diets, however, did not affect the litter size or the birth weight of pups. The DCP intake usually increased with decreases in dietary energy but apparently this did not affect the performance of the mice. It was concluded that an optimal diet should have an MEn value of 3.73 kcal/g DM or more for both the one week post weaning growth period and during lactation, but a diet with an MEn value of 2.86 kcal/g DM was sufficient for growth after 4 weeks of age and during gestation.     

Anti-thyroid antibodies in patients with hyperprolactinemia.

To clarify the possible role of prolactin in the regulation of immune responses in man, we measured circulating anti-thyroid antibodies in 172 normal subjects, 84 patients with prolactinoma and 63 patients with acromegaly with normal thyroid and adrenocortical functions. Frequencies of positive thyroidal microsome and thyroglobulin antibodies were significantly (p < 0.05 and p < 0.01, respectively) higher in women with prolactinoma (20.6% and 20.6%) than in normal women (7.5% and 4.7%). Men with prolactinoma had a significantly (p < 0.05) higher frequency of positive thyroglobulin antibody (18.8%) than normal men (1.5%). When the subjects were divided by decade, women with prolactinoma in the 4th decade had significantly (p < 0.05) higher frequencies of positive thyroidal microsome and thyroglobulin antibodies (30.8% and 30.8%) than normal women of corresponding age (3.7% and 3.7%). In contrast, there was no significant difference in the frequencies of positive anti-thyroid antibodies in patients with acromegaly and in normal subjects. Analysis of the peripheral lymphocyte population revealed that patients with prolactinoma had a higher percentage of B cells than normal subjects, while there was no significant difference in the percentages of total T lymphocytes or in the helper and suppressor T cell ratios in the two groups of subjects. These results suggest that prolactin regulates humoral immune responses in man directly by stimulating B lymphocytes or indirectly by inhibiting suppressor T lymphocyte activity.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

Effects of chocolate containing <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NTM048 on immune function: a randomized,double-blind,placebo-controlled trial

Background

Previous reports showed that oral administration of Leuconostoc mesenteroides strain NTM048 increases IgA levels and CD4+ T cell population in feces and mice, respectively, as revealed by flow cytometric analysis of splenocytes. This study aimed to evaluate the effect of chocolate supplemented with L. mesenteroides strain NTM048 (>?1.00?×?10⁹?CFU/day, NTM048) on the immune parameters of healthy subjects, using a randomized, placebo-controlled, double-blinded study design.

Methods

Participants (mean age: 46.3?years) ingested 28?g of test food daily, at a time of their own choice, for 4?weeks. The immunological parameters of all participants were evaluated two times (pre- and post- ingestion). At the end of the study, various immunological parameters of the participants were measured and scoring of immunological vigor (SIV) was performed using a comprehensive algorithm.

Results

Ingestion of NTM048-supplemented chocolate significantly improved SIV in the NTM048 group (18.6?±?1.6) compared to that in the placebo group (17.8?±?2.0) after 4?weeks (p?=?0.049). Several immunological parameters (CD8⁺T cells, CD8⁺CD28⁺ T cells, and memory T cells) were significantly elevated in the NTM048 group as compared to the placebo group (all p?<?0.05). In addition, T cell proliferation index at post-ingestion significantly increased compared with that at pre-ingestion in the NTM048 (p?=?0.017) and placebo groups (p?=?0.037), although no differences were observed between the two groups.

Conclusion

Our results suggest that ingestion of chocolate supplemented with NTM048 is effective against the age-related decline in T cell-related immune functions.

Trial registration

UMIN Clinical Trials Registry UMIN000021989. Registered 19 April 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000025321

     

Derivation of Human Trophoblast Stem Cells

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Case Report: A patient with spinocerebellar ataxia type 31 and sporadic Creutzfeldt-Jakob disease

We report a Japanese patient with spinocerebellar ataxia type 31 (SCA31) and sporadic Creutzfeldt-Jakob disease (sCJD). A 52-year-old man developed progressive cognitive impairment after the appearance of cerebellar symptoms. Brain MR diffusion-weighted imaging (DWI) demonstrated a slowly expanding hyperintense lesion in the cerebral cortex. The patient was finally diagnosed as having both SCA31 and sCJD by identification of genetic mutations and by real-time quaking-induced conversion (RT-QUIC) analysis of the cerebrospinal fluid (CSF), respectively. Here, we report the clinical details of this rare combined case, with particular reference to the association between prion protein and the early onset of SCA31.     

Effect of eldecalcitol on articular cartilage through the regulation of transcription factor Erg in a murine model of knee osteoarthritis

Clinical studies have reported an association between low blood levels of 25-hydroxyvitamin D and the progression of osteoarthritis (OA), but the mechanism and effects of vitamin D signaling on articular chondrocytes and cartilage remains unclear. The purpose of this study was to investigate the effects of vitamin D on articular cartilage degeneration using eldecalcitol (ED-71), which is an active vitamin D₃ analog. Eight-week old male C57BL/6NCrSlc mice were subjected to experimental surgery to induce OA and local treatments with 10 μL ED-71 (0.5 μg/mL) were administered weekly. Four and 12 weeks after surgery, joints were evaluated using histological scoring systems. In addition, gene expression was analyzed in chondrocytes that were isolated from wildtype neonatal mice, cultured, and treated with ED-71 (10^?8 M). Joints treated with ED-71 demonstrated slowed progression of OA at 4 weeks after surgery, but few effects were observed at 12 weeks after surgery. Ets-related gene (Erg) expression was upregulated in OA articular cartilage, and further increased by ED-71 treatment. In primary chondrocytes cultured with ED-71, the gene expression of Erg and lubricin/proteoglycan 4 significantly increased, as compared to that of cells cultured without ED-71. Local treatment with ED-71 reduced degenerative changes to the articular cartilage during the early phase of experimental OA. Regulation of Erg by ED-71 in articular cartilage could confer resistance to early osteoarthritic changes.     

Reduction of sooty mold damage through biocontrol of the greenhouse whitefly <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> (Hemiptera: Aleyrodidae) using selective insecticides in tomato cultivation greenhouses

The proliferation of sooty mold on tomato fruit, Solanum lycopersicum L.—as caused by the secretion of honeydew on the fruit by greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae)—has recently become a serious problem in estivo-autumnal greenhouse tomato cultivation in Japan. It is becoming increasingly difficult to control T. vaporariorum using insecticides because whiteflies have developed resistance to a variety of insecticides. As the first step towards integrated pest management, we examined whether the use of selective insecticides could prompt a cascade process in which an increase in parasitoids is followed by a decrease in whitefly occurrence and then a reduction in sooty mold damage. We compared greenhouses in which nonselective insecticides effective against T. vaporariorum and its parasitoids were used (hereafter denoted “nonselective insecticide greenhouses”) with greenhouses in which selective insecticides for T. vaporariorum were used (hereafter denoted “selective insecticide greenhouses”) in terms of the density and level of parasitism of T. vaporariorum as well as the degree of damage from sooty mold. The number of parasitized whiteflies increased with the number of whiteflies in the selective insecticide greenhouses, whereas it remained at low levels regardless of the number of whiteflies in the nonselective insecticide greenhouses. Furthermore, the selective insecticide greenhouses showed significantly higher parasitism levels, fewer whiteflies, and reduced sooty mold damage compared to the nonselective insecticide greenhouses. These results suggest that the use of selective insecticides causes an increase in parasitism, which in turn suppresses the number of greenhouse whiteflies and, eventually, sooty mold.