Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Association between Maternal Exposure to di(2-ethylhexyl) Phthalate and Reproductive Hormone Levels in Fetal Blood: The Hokkaido Study on Environment and Children's Health

Prenatal di(2-ethylhexyl) phthalate (DEHP) exposure can produce reproductive toxicity in animal models. Only limited data exist from human studies on maternal DEHP exposure and its effects on infants. We aimed to examine the associations between DEHP exposure in utero and reproductive hormone levels in cord blood. Between 2002 and 2005, 514 pregnant women agreed to participate in the Hokkaido Study Sapporo Cohort. Maternal blood samples were taken from 23–35 weeks of gestation and the concentration of the primary metabolite of DEHP, mono(2-ethylhexyl) phthalate (MEHP), was measured. Concentrations of infant reproductive hormones including estradiol (E2), total testosterone (T), and progesterone (P4), inhibin B, insulin-like factor 3 (INSL3), steroid hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone were measured from cord blood. Two hundred and two samples with both MEHP and hormones'' data were included in statistical analysis. The participants completed a self-administered questionnaire regarding information on maternal characteristics. Gestational age, birth weight and infant sex were obtained from birth records. In an adjusted linear regression analysis fit to all study participants, maternal MEHP levels were found to be associated with reduced levels of T/E2, P4, and inhibin B. For the stratified analyses for sex, inverse associations between maternal MEHP levels T/E2, P4, inhibin B, and INSL3 were statistically significant for males only. In addition, the MEHP quartile model showed a significant p-value trend for P4, inhibin B, and INSL3 decrease in males. Since inhibin B and INSL3 are major secretory products of Sertoli and Leydig cell, respectively, the results of this study suggest that DEHP exposure in utero may have adverse effects on both Sertoli and Leydig cell development in males, which agrees with the results obtained from animal studies. Comprehensive studies investigating phthalates'' exposure in humans, as well as their long-term effects on reproductive development are needed.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Development of an enzyme-linked immunosorbent assay system for the determination of asymmetric dimethylarginine using a specific monoclonal antibody

We produced a monoclonal antibody (mAb) against N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM-100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Dynamics of methane in mesotrophic Lake Biwa,Japan

As a part of a core project of IGBP (International Geosphere-Biosphere Programme), distribution, production, oxidation and transport processes of methane in bottom sediments and lake water in a mesotrophic lake (Lake Biwa) have been studied with special reference to the spatial heterogeneity of each process. In this study, we attempted to synthesize previously reported results with newly obtained ones to depict the methane dynamics in the entire lake. The pelagic water column exhibited subsurface maxima of dissolved methane during a stratified period. Transect observation at the littoral zone suggested that horizontal transportation may be a reason for the high methane concentration in epilimnion and thermocline at the offshore area. Tributary rivers and littoral sediments were suggested to be the source. Observations also showed that the internal wave caused resuspension of the bottom sediment and release of methane from the sediment into the lake water. The impact of the internal waves was pronounced in the late stage of a stratified period. The littoral sediment showed much higher methanogenic activity than the profundal sediments, and the bottom water of the littoral sediments had little methanotrophic activity. In the profundal sediment, most of the methane that diffused up from the deeper part was oxidized when it passed through the oxic layer. Active methane oxidation was also observed in the hypolimnetic water, while the lake water in the epilimnion and thermocline showed very low methane oxidation, probably due to the inhibitory effect of light. These results mean a longer residence time for methane in the epilimnion than in the hypolimnion. Horizontal inflow of dissolved methane from the river and/or littoral sediment, together with the longer residence time in the surface water, may cause the subsurface maxima, which have also been observed in other lakes and in the ocean.     

Geographic variation in the growth of white croaker,Pennahia argentata,off the coast of northwest Kyushu,Japan

We examined geographic variation in the growth of white croaker,Pennahia argentata, off the coast of northwest Kyushu, Japan, Ariake Sound, Tachibana Bay, Omura Bay and the Goto Sea by examination of otoliths. The outer margins of the otoliths showed that a opaque zone was formed once a year, with its peak in June, and could be used as an annulus. White croaker caught during this study reached a maximum age of 10years in the Goto Sea. The growth curves for both sexes in all localities were expressed by the von Bertalanffy growth equations from back-calculated total length of fish. We found significant sexual differences in growth curves in Ariake Sound, Tachibana Bay and the Goto Sea. For both sexes, white croaker in the Goto Sea reached the largest length at each estimated age of the four localities. The growth curves for both sexes showed significant differences among four localities, suggesting that several stocks may exist in the study area although the greatest distance between each locality was at most 30 km.