Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

{beta}-1,4-Glucan occurring in homogenate of Phaseolus aureus seedlings. Possible nascent stage of cellulose biosynthesis in vivo

A small amount of cytoplasmic ß-1,4-glucan, whichmight be involved in the synthesis of cellulose in the cellwall, was found in the homogenate prepared from the hypocotylsof seedlings of Phaseolus aureus. Upon hydrolysis by cellulaseof the 20,000?g pellet from the cytoplasmic fraction of segmentsincubated in a [¹⁴C]-glucose solution, [¹⁴C]-cellobiose wasproduced, with specific radioactivities 3 to 10 times greaterthan those of the cellobiose from cellulose in the cell wallat various incubation periods. The incoporation of radioactivityfrom [¹⁴C]-glucose into this cytoplasmic ß-1,4-glucanwas therefore faster than that into cellulose constituting thecell wall. Hence, it seemed that the former ß-1,4-glucancould be turned over. To examine whether the- cytoplasmic ß-1,4-glucanis carried by some subcellular components, cytoplasmic ß-1,4-glucanin the cell was fractionated by differential centrifugation,two enzyme activities being measured as the markers of subcellularcomponents. The distribution of ß-1,4-glucan was similarto that of UDPG-glucosyltransferase activity but not to thatof IDP-ase activity. The result suggests that the cytoplasmicß-1,4-glucan has some relation to plasma membranes. Coumarin, known as a specific inhibitor for the biosynthesisof cellulose in plant cells, was shown to inhibit the incorporationof radiocarbon from [¹⁴C]-glucose into cytoplasmic ß-1,4-glucanto the same extent as that into cellulose in the cell wall ofthe hypocotyls. ¹ Present address: Department of Biological Science, TohokuUniversity, Kawauchi, Sendai 980, Japan. (Received May 31, 1976; )     

Degradation of Xyloglucan by Wall-bound Enzymes from Soybean Tissue I. Occurrence of Xyloglucan-degrading Enzymes in Soybean Cell Wall

An enzyme preparation that catalyzes the degradation of xyloglucanwas obtained by extraction of the cell walls of soybean hypocotylswith a buffer containing 1.0 M NaCl. The enzyme preparationwas shown to catalyze two-step degradation of xyloglucan. Thepolysaccharide was first degraded into comparatively large fragments,which were then further degraded into monosaccharides. In orderto elucidate the mode of degradation of the xyloglucan duringcell growth, the activities of xyloglucandegrading enzymes ofsoybean-hypocotyl segments were assayed at different stagesof elongation. The total activities of the degrading enzymeswere lower in the elongating regions than in the non-elongatingregions. However, high levels of endo-ß-l,4-glucanasewere found in the elongating regions. These results suggestthat xyloglucan is hydrolyzed by endo-ß-1,4-glucanaseinto comparatively large fragments at the initial stage of growthand the resulting fragments are further degraded into monosaccharidesduring cell elongation. (Received May 20, 1981; Accepted August 8, 1981)     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

Siblings with renal tubular acidosis and nerve deafness. The first family in Japan

Summary Two siblings with renal tubular acidosis (RTA) and nerve deafness were examined. It was found by ammonium chloride and bicarbonate loading tests that the 6-year-old brother had a hybrid type of RTA and his 4-year-old sister, a distal type of RTA. Enzyme activity and amount of enzyme protein of carbonic anhydrase isoenzyme I and II in red blood cells, measured using an immunoadsorbent method, were normal in both cases. Although this indicated that the RTAs of these patients are not generated by the carbonic anhydrase deficiency, an investigation with renal tissue is necessary to arrive at a final conclusion.     

Low-serum culture system improves the adipogenic ability of visceral adipose tissue-derived stromal cells

In obese adipose tissue, infiltrating macrophages release proinflammatory cytokines that trigger insulin resistance. An adipocyte-based platform from visceral fat would be useful to elucidate the pathology of adipose inflammation and to develop therapeutic drugs for insulin resistance. ADSCs (adipose tissue-derived mesenchymal stromal cells) expanded from subcutaneous fat are intensively studied as sources for regenerative medicine. However, the adipocyte culture system from visceral fat tissue has not been utilized yet. We aimed to establish the bioactive adipocyte platform using ADSCs from visceral fat pad. Stromal vascular fractions were processed from epididymal fat pads of Sprague-Dawley rats and three human omental fat pads, and the ADSCs were expanded using a low-serum culture method. The responses of ADSCs and ADSC-adipocytes (their adipogenic lineages) to pioglitazone, a therapeutic drug for diabesity, were evaluated by gene expression and ELISA. ADSCs (1×108) were expanded from 10 g of rat epididymal fat pads or human omental fat pads over five passages. Cell surface marker expressions revealed that visceral ADSCs were equivalent to mesenchymal stem cells. ADSC-adipocytes expanded in the low-serum culture system significantly showed higher expression of adipogenic markers [PPAR (peroxisome proliferator-activated receptor) γ, LPL (lipoprotein lipase) and FABP4 (fatty acid-binding protein 4)] and adipocytokines [adiponectin, resistin, leptin, PAI-1 (plasminogen-activator inhibitor 1) and IL (interleukin)-10] than those expanded in a high-serum culture system. Pioglitazone accelerated the adipogenic induction and increased adiponectin expression in human ADSCs by 57.9±5.8-fold (mean±S.E.M.) relative to control cells (P<0.001). Both in rat and human ADSC-adipocytes, TNF-α significantly induced proinflammatory cytokines [MCP-1 (monocyte chemoattractant protein-1) and IL-6] and suppressed adiponectin expression, while pioglitazone antagonized these effects. The present findings suggest that visceral ADSC-adipocytes expanded in low-serum culture would be useful for adiposcience and pharmacological evaluations.     

Changes of aliphatic C–H bonds in cyanobacteria during experimental thermal maturation in the presence or absence of silica as evaluated by FTIR microspectroscopy

Aliphatic C–H bonds are one of the major organic signatures detected in Proterozoic organic microfossils, and their origin is a topic of interest. To investigate the influence of the presence of silica on the thermal alteration of aliphatic C–H bonds in prokaryotic cells during diagenesis, cyanobacteria Synechocystis sp. PCC6803 were heated at temperatures of 250–450°C. Changes in the infrared (IR) signals were monitored by micro‐Fourier transform infrared (FTIR) spectroscopy. Micro‐FTIR shows that absorbances at 2,925 cm^?1 band (aliphatic CH₂) and 2,960 cm^?1 band (aliphatic CH₃) decrease during heating, indicating loss of the C–H bonds, which was delayed by the presence of silica. A theoretical approach using solid‐state kinetics indicates that the most probable process for the aliphatic C–H decrease is three‐dimensional diffusion of alteration products under both non‐embedded and silica‐embedded conditions. The extrapolation of the experimental results obtained at 250–450°C to lower temperatures implies that the rate constant for CH₃ (k_CH3) is similar to or lower than that for CH₂ (k_CH2; i.e., CH₃ decreases at a similar rate or more slowly than CH₂). The peak height ratio of 2,960 cm^?1 band (CH₃)/2,925 cm^?1 band (CH₂; R_3/2 values) either increased or remained constant during the heating. These results reveal that the presence of silica does affect the decreasing rate of the aliphatic C–H bonds in cyanobacteria during thermal maturation, but that it does not significantly decrease the R_3/2 values. Meanwhile, studies of microfossils suggest that the R_3/2 values of Proterozoic prokaryotic fossils from the Bitter Springs Group and Gunflint Formation have decreased during fossilization, which is inconsistent with the prediction from our experimental results that R_3/2 values did not decrease after silicification. Some process other than thermal degradation, possibly preservation of specific classes of biomolecules with low R_3/2 values, might have occurred during fossilization.     

Functional analysis of chick heparan sulfate 6‐O‐sulfotransferases in limb bud development

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O‐sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O‐sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS‐6‐O‐sulfotransferases (HS6ST‐1 and HS6ST‐2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf‐8 and Fgf‐10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST‐2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf‐8 and Fgf‐10 expressions than that achieved with HS6ST‐1 RNAi. HS6ST‐1 RNAi and HS6ST‐2 RNAi caused a significant but distinct reduction in the levels of different 6‐O‐sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6‐O‐sulfation of HS play essential roles in chick limb bud development.     

Absolute stereostructure of potent alpha-glucosidase inhibitor, Salacinol, with unique thiosugar sulfonium sulfate inner salt structure from Salacia reticulata

A most potent alpha-glucosidase inhibitor named salacinol has been isolated from an antidiabetic Ayurvedic traditional medicine, Salacia reticulata WIGHT, through bioassay-guided separation. The absolute stereostructure of salacinol was determined on the basis of chemical and physicochemical evidence, which included the alkaline degradation of salacinol to 1-deoxy-4-thio-D-arabinofuranose and the X-ray crystallographic analysis, to be the unique spiro-like configuration of the inner salt comprised of 1-deoxy-4-thio-D-arabinofuranosyl sulfonium cation and 1'-deoxy-D-erythrosyl-3'-sulfate anion. Salacinol showed potent inhibitory activities on several alpha-glucosidases, such as maltase, sucrase, and isomaltase, and the inhibitory effects on serum glucose levels in maltose- and sucrose-loaded rats (in vivo) were found to be more potent than that of acarbose, a commercial alpha-glucosidase inhibitor.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.