Cooperative activity of multiple upper layer proteins for thalamocortical axon growth

During development, sensory thalamocortical (TC) axons grow into the neocortex and terminate primarily in layer 4. To study the molecular mechanism that underlies lamina-specific TC axon termination, we investigated the responsiveness of TC axons to ephrin-A5, semaphorin-7A (Sema7A) and kit ligand (KL), which are expressed in the upper layers of the developing cortex. Dissociated cells of the dorsal thalamus from embryonic rat brain were cultured on dishes that were coated with preclustered Fc-tagged extracellular domains of these molecules. Each protein was found to promote TC axon growth in a dose-dependent fashion of a bell-shaped curve. Any combination of the three proteins showed a cooperative effect in lower concentrations but not in higher concentrations, suggesting that their growth-promoting activities act in a common pathway. The effect of spatial distributions of these proteins was further tested on a filter membrane, in which these proteins were printed at a size that recapitulates the scale of laminar thickness in vivo, using a novel protein-printing technique, Simple-To-mAke Micropore Protein-Printing (STAMP2) method. The results demonstrated that TC axons grew massively on the laminin-coated region but were prevented from invading the adjacent ephrin-A5-printed region, suggesting that TC axons detect relative differences in the growth effect between these regions. Moreover, the inhibitory action of ephrin-A5 was enhanced by copresence with KL and Sema7A. Together, these results suggest that the lamina-specific TC axon targeting mechanism involves growth-inhibitory activity by multiple molecules in the upper layers and detection in the molecular environments between the upper and deep layers.     

Molecular survey of Babesia microti, Ehrlichia species and Candidatus neoehrlichia mikurensis in wild rodents from Shimane Prefecture, Japan

A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.     

ESA Hyporesponsiveness Is Associated with Adverse Events in Maintenance Hemodialysis (MHD) Patients,But Not with Iron Storage

Objective

It has been reported that hyporesponsiveness to erythropoiesis-stimulating agent (ESA) is associated with adverse events in patients on maintenance hemodialysis (MHD). However, it has not been determined whether higher iron storage is associated with an improved response, including better survival, to ESA.

Design and Method

We measured serum ferritin, hemoglobin (Hb), and transferrin saturation (TSAT) levels every three months for two years in 1,095 MHD patients. The weekly dose of ESA to Hb ratio was also calculated as an index of ESA responsiveness (ERI).

Results

A significant correlation (p<0.001, R = 0.89) between ferritin and Hb was only observed in the patients with ferritin levels <50 ng/mL. High-dose (≥50 mg/week) intravenous iron administration, female sex, low serum albumin, and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker use were significant predictors of a high ERI value (>280); however, serum ferritin and TSAT levels did not predict a higher ERI. In the time-dependent Cox hazard model, the risk for a composite event in the patients with a high ERI (≥280) and a high ferritin level (≥100 ng/mL) was significantly greater (hazard ratio [HR], 2.09, P = 0.033) than that for patients with a high ERI and a low ferritin (<100 ng/mL) level.

Conclusion

Hb was dependent upon ferritin levels in patients with ferritin levels <50 ng/mL but not in patients with ferritin levels ≥50 ng/mL. Patients with hyporesponsiveness to ESA had a greater risk of composite events, but ERI was unrelated to iron storage.     

The levels of main urinary metabolite of prostaglandin F1α and F2α in human subjects measured by radioimmunoassay

Radioimmunoassay technique for measuring 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, the main urinary metabolite of PGF₁α and PGF₂α (PGF₂α-MUM), was further improved.It was postulated based on some experimental data that the PGF₂α-MUM exists in the urine mostly as dioic acid form, not as δ-lactone formThe daily excretion of PGF₂α-MUM in men ranged from 14.43 μg to 36.14 μg and in women from 5.21 μg to 14.25 μg.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.     

Gonadotropic regulation of circadian clockwork in rat granulosa cells

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.     

Evaluation of the permeability of hair growing ingredient encapsulated PLGA nanospheres to hair follicles and their hair growing effects

This paper describes the process of encapsulating hair growing ingredients in the PLGA nanospheres by emulsion solvent diffusion method and investigates the feasibility of using the PLGA nanospheres as the DDS (Drug delivery System) carriers for delivering various hair growing ingredients to hair follicles. In-vitro and in-vivo tests were conducted to verify the performances of encapsulated PLGA nanospheres with three different hair growing ingredients. In the in-vitro tests, the scalp-pore permeability of hair growing ingredient encapsulated PLGA nanospheres (dispersed in the PBS solution) was examined using human scalp biopsies in a modified Bronaugh diffusion chamber in comparison to that of the control samples containing the hair growing ingredient in the PBS solution. Furthermore, the hair growing effect of the encapsulated PLGA nanospheres was evaluated with the C3H mice in the in-vivo tests. By observing the fluorescence intensity of the ingredients, as shown in the cross-section photographs of the human scalp biopsies, it was found that the dispersion liquids containing hair growing ingredient encapsulated PLGA nanospheres exerted a scalp-pore permeability 2.0- to 2.5-fold more marked than that of the control samples. Also, the hair growing activities were enhanced by using the encapsulated PLGA nanospheres, which transformed the hair growth cycle from the resting phase to the growing phase. As a result, the degree of hair growth was improved significantly. These results suggested that the PLGA nanosphere can be a new DDS carrier for delivering hair growing ingredients and drugs to the hair follicles.